Methods for enriching alpha-tocotrienol from mixed tocol compositions

ABSTRACT

Provided herein are methods for making an alpha-tocotrienol enriched tocol mixture, such as from a plant or plant-derived material. Also provided herein are simulated moving bed purification methods for tocotrienol compounds such as alpha tocotrienol.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/063,035, filed Jun. 15, 2018 (now U.S. Pat. No. 10,745,371, issuedAug. 18, 2020), which is a filing under 35 U.S.C. § 371 of InternationalPatent Application No. PCT/US2016/067377, filed Dec. 16, 2016, whichclaims the benefit of U.S. Provisional Patent Application No.62/386,943, filed Dec. 16, 2015, and of U.S. Provisional PatentApplication No. 62/386,949, filed Dec. 16, 2015. Each of the foregoingapplications is incorporated herein by reference in its entirety.

FIELD

Provided herein are methods for making an alpha-tocotrienol enrichedtocol mixture, such as from a plant or plant-derived material. Alsoprovided herein are simulated moving bed purification methods fortocotrienol compounds such as alpha tocotrienol.

BACKGROUND

Certain plants contain mixtures of tocol compounds, such as tocopherol(alpha, beta, gamma and/or delta tocopherols) and/or tocotrienol (alpha,beta, gamma and/or delta tocotrienols) compounds. Previous methods havebeen used to obtain alpha-tocotrienol containing compositions fromnatural plant sources, such as palm oils and refined palm oils. Someprocesses for producing alpha-tocotrienol from plant sources producealpha-tocotrienol by alkylating beta-, gamma-, and delta-tocotrienols;however, those processes result in a lower amount of alpha-tocotrienolthan might otherwise be obtained. In particular, it has been difficultto obtain desirable amounts of alpha-tocotrienol from plant sourcescontaining more than about 5% phytosterol content.

What is needed are improved methods for enriching alpha-tocotrienol fromnatural plant sources. Furthermore, what is needed are methods forenriching alpha-tocotrienol from natural plant sources, wherein higheramounts of alpha-tocotrienol may be obtained by converting the beta-,gamma-, and delta-tocotrienols to alpha-tocotrienol. The methodsprovided herein are capable of solving this and other problems.

SUMMARY

In one aspect is a method of making an alpha-tocotrienol enriched tocolmixture. In some embodiments, the method includes (a) contacting a tocolmixture with an amino-alkylating agent, wherein the tocol mixturecomprises at least one non-alpha tocotrienol, at least one non-tocol,optionally alpha tocotrienol, and optionally one or more tocopherols,whereby the at least one non-alpha tocotrienol is amino-alkylated; (b)reducing the amino-alkylated non-alpha tocotrienols to alpha tocotrienolwith a reducing agent; (c) removing one or more waxy impurities from themixture; (d) contacting the mixture with an agent that binds one or morepolar impurities; and (e) removing the agent. In some embodiments, themethod further includes after step (c): (c)(1) converting thealpha-tocotrienol to an alpha-tocotrienol salt; (c)(2) providing a polarsolvent phase and a non-polar solvent phase and partitioning thealpha-tocotrienol salt to the polar solvent phase; (c)(3) removing thenon-polar solvent phase; and (c)(4) converting the alpha-tocotrienolsalt in the polar solvent phase to alpha-tocotrienol. In someembodiments, including any of the foregoing embodiments, the methodfurther includes after step (a): (a)(1) removing water formed from thereaction of step (a).

In some embodiments, the alpha-tocotrienol in step (c)(1) is convertedto the alpha tocotrienol salt with an inorganic salt base having apK_(a) of more than about 10. In some embodiments, the inorganic saltbase is sodium methoxide (NaOMe).

In some embodiments, the polar solvent phase in step (c)(2) containsN-methyl-pyrrolidone (NMP). In some embodiments, the non-polar solventphase in step (c)(2) contains an alkane. In some embodiments, thenon-polar solvent phase in step (c)(2) contains a hexane or a heptane.In some embodiments, the non-polar solvent phase in step (c)(2) containsn-heptane.

In some embodiments, the partitioning between the polar and non-polarsolvent phases in step (c)(2) is performed at less than 10° C. In someembodiments, the partitioning between the polar and non-polar solventphases in step (c)(2) is performed at about 0° C.

In some embodiments, converting the alpha tocotrienol salt to alphatocotrienol in step (c)(4) includes addition of an acid having a pKa ofless than about 7. In some embodiments, converting the alpha tocotrienolsalt to alpha tocotrienol in step (c)(4) includes addition of HCl.

In some embodiments, step (c)(4) includes production of a saltby-product, and the method further includes a step (c)(5) after step(c)(4), wherein step (c)(5) includes removing the salt by-productproduced in step (c)(4). In some embodiments, step (c)(5) comprisessolvent extraction with a non-polar light phase; followed by retainingthe non-polar light phase. In some embodiments, the non-polar lightphase comprises toluene, a heptane or a combination thereof. In someembodiments, the heptane is n-heptane. In some embodiments, thenon-polar light phase is washed with an aqueous wash.

In some embodiments, step (a)(1) includes an azeotropic distillation toremove the water. In some embodiments, step (a)(1) includes anazeotropic distillation in the presence of a solvent with a boilingpoint of at least 80° C. at 1 atm pressure. In some embodiments, step(a)(1) includes an azeotropic distillation in the presence of a solventwith a boiling point of at least 95° C. at 1 atm pressure. In someembodiments, the solvent is selected from the group consisting of:2-butanol, 2-pentanol; 2-methyl-2-butanol, ethanol, toluene, ethanol,toluene, ethyl acetate, acetonitrile, methyl ethyl ketone, cyclohexanol,2-pentanol, 2-hexanol, and 2-methyl-1-propanol. In some embodiments, thesolvent is 2-methyl-2-butanol.

In some embodiments, the amino-alkylating agent in step (a) includes asecondary amine and a formaldehyde equivalent. In some embodiments, thesecondary amine is N-methyl-piperazine. In some embodiments, theformaldehyde equivalent is paraformaldehyde.

In some embodiments, the reducing agent in step (b) is a borohydride. Insome embodiments, the reducing agent in step (b) is NaBH₃CN. In someembodiments, an alcohol having a boiling point of at least about 95° C.is used as a solvent in step (b). In some embodiments, the solvent instep (b) is a four or five carbon alcohol. In some embodiments, thesolvent in step (b) is 2-methyl-2-butanol.

In some embodiments, wherein one or more byproducts are produced insteps (a) and/or (b), and wherein the mixture optionally containsresidual secondary amine, the method includes a step (b)(1) after step(b), wherein step (b)(1) includes removing at least one of the one ormore byproducts produced in steps (a) and/or (b) and removing theoptional residual secondary amine. In some embodiments, step (b)(1)includes an aqueous work up. In some embodiments, the aqueous work upincludes (i) contacting the mixture with isopropyl acetate and water,whereby an aqueous phase is formed; (ii) removing the aqueous phase; and(iii) performing an acid wash on the mixture. In some embodiments, step(i) of the aqueous work up includes contacting the mixture withisopropyl acetate, water, and Na₂HPO₄, whereby an aqueous phase isformed. In some embodiments, the aqueous work up comprises (i)contacting the mixture with isopropyl acetate and water, whereby anaqueous phase is formed; (ii) removing the aqueous phase; (iii)contacting the mixture with water, whereby an aqueous phase is formed,and removing the aqueous phase, and (iv) performing an acid wash on themixture.

In some embodiments, step (c) includes cooling and/or removing solventthat may be present in the mixture to precipitate the one or more waxyimpurities. In some embodiments, the cooling in step (c) includescooling to about −35° C. to about 5° C. In some embodiments, the coolingin step (c) includes cooling to about −20° C. to about −10° C. In someembodiments, the cooling in step (c) comprises cooling to about −35° C.to about −15° C.

In some embodiments, removing the one or more waxy impurities in step(c) includes contacting the mixture and a solid binding material. Insome embodiments, wherein removing the one or more waxy impuritiescomprises contacting the mixture with the solid binding material toproduce a slurry, followed by filtering the slurry and retaining thefiltrate. In some embodiments, the solid binding material is a finelydivided solid comprising carbon, silica, alumina, or diatomaceous earth.In some embodiments, the solid binding material is Celite®.

In some embodiments, step (c) includes addition of heptane and holdingthe mixture at a temperature of about 20° C. to 30° C. In someembodiments, the mixture is held at a temperature of about 20° C. to 30°C. for from about 1 to about 4 hours. In some embodiments, step (c)further includes filtering the mixture to remove insoluble solidby-products. In some embodiments, step (c) includes an aqueous work up.In some embodiments, the aqueous work up comprises (i) contacting themixture with water, whereby an aqueous phase is formed, and removing theaqueous phase, and (ii) performing an acid wash on the mixture. In someembodiments, the acid wash comprises citric acid.

In some embodiments, step (c) includes reducing the mixture to about aminimum stir volume. In some embodiments, the mixture is reduced to aminimum stir volume by distillation in the presence of a solvent. Insome embodiments, the solvent is heptane, 1-AmOH, or a combinationthereof.

In some embodiments, the agent in step (d) comprises silica, Celite®,Diatomaceous earth, alumina, or florisil. In some embodiments, steps (d)and (e) comprise adding the mixture to a column comprising the agent,and eluting the mixture off the column.

In some embodiments, the method further includes after step (e): (f)solvent exchange of the mixture to a solvent selected from the groupconsisting of methanol and acetonitrile. In some embodiments, thesolvent exchange is to methanol. In some embodiments, the solventexchange is to acetonitrile. In some embodiments, the solvent exchangeis carried out in the absence of light.

In some embodiments, the tocol mixture in step (a) is a palm oil or amaterial derived from palm oil. In some embodiments, the tocol mixtureis Tocomin 50®. In some embodiments, the phytosterol content of thetocol mixture is at least about 5%, at least about 7%, at least about8%, at least about 10%, or at least about 12% In some embodiments, thetocol mixture in step (a) comprises at least about 65 (A) % tocols, atleast about 68 (A) % tocols, or at least about 70 (A) % tocols. In someembodiments, the tocol mixture is Gold Tri.E™ 70. In some embodiments,the tocol mixture comprises 84 (A) % of a mixture of tocotrienols andtocopherols.

In some embodiments, the product of the method has a content of at leastabout 50 wt % alpha-tocotrienol, at least about 55 wt. %alpha-tocotrienol, or at least about 58 wt % alpha-tocotrienol.

In another aspect is a method of purifying a tocotrienol feed solution,comprising passing the feed solution through a chromatographic process.In some embodiments, the chromatographic process is a simulated movingbed (SMB) apparatus. In some embodiments, the method includes: (a)passing the feed solution through a chromatographic process comprising astationary phase and a mobile phase stream, wherein said feed solutioncomprises the tocotrienol and one or more impurity compounds; (b)operating the chromatographic process as a simulated moving bed (SMB)process under conditions effective to purify the tocotrienol from atleast one impurity compound; (c) collecting a stream from the SMBapparatus, wherein said stream comprises the purified tocotrienol; and(d) optionally repeating steps (a)-(c).

In some embodiments, the chromatographic process is a continuouschromatographic process. In some embodiments, the chromatographicprocess is a semi-continuous chromatographic process.

In some embodiments, the stream containing the purified tocotrienol isthe extract stream. In some embodiments, the stream containing thepurified tocotrienol is the raffinate stream.

In some embodiments, the stream containing the purified tocotrienol isthe extract stream in a first pass, and wherein the stream containingthe purified tocotrienol is the raffinate stream in a second orsubsequent pass. In some embodiments, the stream containing the purifiedtocotrienol is the raffinate stream in a first pass, and wherein thestream containing the purified tocotrienol is the extract stream in asecond or subsequent pass.

In some embodiments, the tocotrienol that is purified is selected fromthe group consisting of alpha-tocotrienol, beta-tocotrienol,delta-tocotrienol, and-gamma tocotrienol. In some embodiments, thetocotrienol that is purified is alpha-tocotrienol.

In some embodiments, the stationary phase is selected from the groupconsisting of silica gel, functionalized silica gel, reverse phase gel,or chiral phase gel. In some embodiments, the stationary phase has aparticle size of about 2 to about 300 μm, about 5 to about 50 μm, orabout 20 to about 30 μm.

In some embodiments, the mobile phase stream includes one or moresolvents selected from the group consisting of: water, acetonitrile,t-AmOH, methanol, ethanol, n-proposal, isopropyl alcohol, butanol, ethylacetate, isopropyl acetate, MtBE, diethyl ether, fluorinated solvents,alkanes, hexanes, n-hexane, heptanes, n-heptane, methyl-cyclopentane,pentane, methyl-cyclohexane, cyclohexane, Toluene, and CO₂. In someembodiments, the mobile phase stream contains acetonitrile. In someembodiments, the mobile phase stream contains methanol.

In some embodiments, the tocotrienol feed solution is obtained using amethod of making an alpha-tocotrienol enriched tocol mixture asdisclosed herein.

In some embodiments, the a final product stream containing purifiedtocotrienol contains the tocotrienol with at least about 90 (A) %purity. In some embodiments, the final product stream containingpurified tocotrienol is concentrated to a tocotrienol concentration ofat least about 90 wt %.

In some embodiments, the chromatographic process includes 2 to 30columns serially connected, 2 to 12 columns serially connected, or 5 to8 columns serially connected.

In some embodiments, the chromatographic process is operated at a rateof about 0.05 to about 5 kg feed stream per kg stationary phase per 24hours, or about 1 to about 3 kg feed stream per kg stationary phase per24 hours. In some embodiments, the chromatographic process is operatedat a pressure of about 2 bar to about 100 bar, about 5 bar to about 60bar, 20 bar to about 45 bar, or about 30 bar to about 45 bar pressure.In some embodiments, the chromatographic process is operated at atemperature of about 10° C. to about 50° C., about 15° C. to about 40°C., about 20° C. to about 35° C., or about 25° C. to about 30° C.

In some embodiments, the extract stream is collected and tocotrienolcontaining fractions are subjected to a method of making analpha-tocotrienol enriched tocol mixture as disclosed herein. In someembodiments, the extract stream which is subjected a method of making analpha-tocotrienol enriched tocol mixture as disclosed herein is thenpurified according to a method of purifying a tocotrienol feed solutionas disclosed herein.

In some embodiments, the raffinate stream is collected and tocotrienolcontaining fractions are subjected to a method of making analpha-tocotrienol enriched tocol mixture as disclosed herein. In someembodiments, the raffinate stream which is subjected a method of makingan alpha-tocotrienol enriched tocol mixture as disclosed herein is thenpurified according to a method of purifying a tocotrienol feed solutionas disclosed herein.

In another aspect of the invention is a composition comprisingalpha-tocotrienol, produced by a method as described herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a schematic of an exemplary embodiment of a method ofmaking an alpha-tocotrienol enriched tocol mixture as disclosed herein.

FIG. 2 provides a schematic of an exemplary embodiment of a method ofmaking an alpha-tocotrienol enriched tocol mixture as disclosed herein.Dotted lines indicates steps that are optional, depending on the amountof tocotrienol or alpha-tocotrienol in the initial starting material.

DESCRIPTION OF EXEMPLARY EMBODIMENTS Definitions

It is to be understood that the description of methods and compositionsdescribed herein include “comprising”, “consisting of”, and “consistingessentially of” embodiments. For example, when a method is described as“consisting essentially of” the listed steps, the method contains thesteps listed, and may contain other steps that do not substantiallyaffect the method for producing the alpha-tocotrienol enrichedcompositions, but the method does not contain any other steps whichsubstantially affect the method other than those steps expressly listed.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, and unless otherwise specified, the terms “about” and“approximately,” when used in connection with e.g. temperatures,concentration, amounts, size, pK, pH, or weight percent, mean atemperature, concentration, amount, size, pK, pH, or weight percent thatis recognized by those of ordinary skill in the art to provide anequivalent effect to that obtained from the specified temperature,concentration, amount, size, pK, pH, or weight percent. Specifically,the terms “about” and “approximately,” when used in this context,contemplate a temperature, concentration, amount, size, pK, pH, orweight percent within 15%, within 10%, within 5%, within 4%, within 3%,within 2%, within 1%, or within 0.5% of the specified temperature,concentration, amount, size, pK, pH, or weight percent.

The terms “a” or “an,” as used in herein means one or more, unlesscontext clearly dictates otherwise.

“Amino-alkylating agent” as used herein means an agent capable of addingan amino-alkyl group to a tocotrienol. For example, an amino-alkylatingagent can include a secondary amine and a formaldehyde equivalent,wherein the secondary amine and formaldehyde equivalent are as disclosedherein.

“Tocol” indicates tocopherols and/or tocotrienols, in some embodimentsalpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol,alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and/ordelta-tocotrienol. This includes all stereoisomers of the tocotrienoland tocopherol compounds, including, for example, diastereomers andenantiomers. The term “tocol” also includes mixtures of tocopheroland/or tocotrienol stereoisomers in any ratio, including, but notlimited to, racemic mixtures, and their use in the methods. Unlessstereochemistry is explicitly indicated in a structure, the structure isintended to embrace all possible stereoisomers of the compound depicted.If stereochemistry is explicitly indicated for one portion or portionsof a molecule, but not for another portion or portions of a molecule,the structure is intended to embrace all possible stereoisomers for theportion or portions where stereochemistry is not explicitly indicated.The structures of exemplary tocopherols and tocotrienols is provided inTable 1 below:

TABLE 1

R₁ R₂ R₃ Alpha- tocotrienol

methyl methyl methyl Beta- tocotrienol

methyl H methyl Gamma- tocotrienol

H methyl methyl Delta- tocotrienol

H H methyl Alpha- tocopherol

methyl methyl methyl Beta- tocopherol

methyl H methyl Gamma- tocopherol

H methyl methyl Delta- tocopherol

H H methyl

In some embodiments, exemplary tocopherols and tocotrienols contemplatedfor use in the methods disclosed herein have the structures as providedin Table 2 below:

TABLE 2 Alpha-tocotrienol

Beta-tocotrienol

Gamma-tocotrienol

Delta-tocotrienol

Alpha-tocopherol

Beta-tocopherol

Gamma-tocopherol

Delta-tocopherol

A “tocol mixture” comprises at least one tocol (e.g. one, two, three,four, or more tocols). In certain embodiments, including any of theforegoing embodiments, the tocol mixture is derived from a plantmaterial. In certain embodiments, including any of the foregoingembodiments, the tocol mixture is derived from palm oil, a palm fruitextract, or a mixture of palm oil and palm fruit extract. In someembodiments, the tocol mixture is derived from palm oil. In someembodiments, the tocol mixture is a commercially available productcomprising an enriched tocotrienol extract derived from palm oil. Forexample, the commercially available product can be one or more productsas provided by Carotech, Golden Hope Bioorganic, Davos Life Science,Beijing Gingko Group; Eisai, Eastman Corporation, Oryza Oil & FatCompany, Sime Darby Biorganic Sdn Bhd or Palm Nutraceuticals. Thesecommercially available products include, but are not limited to, NuTriene Tocotrienol® (30% content, a product of Eastman ChemicalCompany); various Oryza® tocotrienol products of different tocotrienolconcentrations from Oryza Oil & Fat Co. Ltd; including Oryzatocotrienol-70 with 70% total tocopherol/tocotrienol content, and atotal tocotrienol content of 40% including 14% of alpha-tocotrienol and24% gamma-tocotrienol, and Oryza tocotrienol-90 with 90% totaltocopherol/tocotrienol content and a total tocotrienol content of 60%;Golden Hope Plantations Berhad Tocotrienol oil (70% content), Davos LifeScience TRF (63% content), Davos Life Science TC84 (84% content);Ginnoway™ tocotrienol concentrate from palm and rice oil from BeijingGingko Group, Gold Tri.E™, a product of Sime Darby Biorganic Sdn Bhd andPalm Nutraceuticals Sdn Bhd (89% content). Delta Tocotrienol-92® (92%pure by HPLC) is a commercially available product from Beijing GingkoGroup that may be also used in the methods disclosed herein. In certainembodiments, the tocol mixture is a Tocomin® product, for example,Tocomin 500. In some embodiments, the tocol mixture is Gold Tri.E™ 70.In some embodiments, the tocol mixture is natural palm oil, e.g., TC84(Davos Life Science).

“Non-tocol” indicates an organic compound other than a tocol. In certainembodiments, including any of the foregoing embodiments, the non-tocolis selected from the group consisting of sterols, tocomonoenols,tocodienols, squalene, carotenoids, and glycerate esters.

“Solvent exchange” indicates replacing a solvent (or solvent mixture)with a different solvent (or solvent mixture). In certain embodiments,including any of the foregoing embodiments, the original solvent isremoved by vacuum distillation. In certain embodiments, including any ofthe foregoing embodiments, less than about 10% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 9% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 8% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 7°/o of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 6% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 5% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 4% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 3% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 2% of the original solventremains after solvent exchange. In certain embodiments, including any ofthe foregoing embodiments, less than about 1% of the original solventremains after solvent exchange.

“Minimum stir volume” indicates the minimum volume of liquid needed inthe reactor so that the stirring mechanism effectively stirs the reactorcontents. For example, minimum stir volume can indicate about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, or 75% of thevolume capacity of the reactor.

“Waxy impurities” indicates one or more undesired organic compounds eachhaving a melting temperature of at least about 25° C. In someembodiments, the undesired organic compounds contain one or morehydrocarbon tails that are at least C₂₀ in length. In some embodiments,the undesired organic compounds contain one or more hydrocarbon tailsthat are at least C₂₂ in length. In some embodiments, the undesiredorganic compounds contain one or more hydrocarbon tails that are atleast C₂₄ in length. In some embodiments, the undesired organiccompounds contain one or more hydrocarbon tails that are at least C₂₆ inlength. In some embodiments, the undesired organic compounds contain oneor more hydrocarbon tails that are at least C₂₈ in length. In someembodiments, the undesired organic compounds contain one or morehydrocarbon tails that are at least C₃₀ in length. Waxy impurities caninclude, for example, squalene and other high molecular weight isoprenecompounds having a melting temperature of at least about 25° C., atleast about 28° C., or at least about 30° C.

“Polar impurities” indicates one or more undesired compounds that aremore polar than the desired alpha-tocotrienol.

“Non-polar impurities” indicates one or more undesired compounds thatare less polar than the desired alpha-tocotrienol.

“Polar solvent” indicates a solvent with significant bond polarizations,typically solvents with heteroatoms, such as ethyl acetate, methanol,acetonitrile. In another embodiment, a polar solvent is a solvent with adielectric constant of greater than 15. The skilled practitioner wouldrecognize whether a particular solvent is classified as polar. In someembodiments, a polar solvent is one or more solvents selected from thegroup consisting of tetrahydrofuran (THF), ethyl acetate (EA),acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water,ethanol, methanol, and isopropanol. The skilled practitioner wouldcontemplate other polar solvents which could be used herein. A polarprotic solvent is one in which a hydrogen atom is bound to an oxygen orto a nitrogen.

“Non-polar solvent” indicates a solvent with very low or no bondpolarizations, typically hydrocarbon solvents, such as heptane. Inanother embodiment, a solvent with a dielectric constant of less than15. The skilled practitioner would recognize whether a particularsolvent is classified as non-polar. In some embodiments; a non-polarsolvent is one or more solvents selected from the group consisting of:dichloromethane, toluene, benzene, 1,4-dioxane, hexane, diethyl ether.The skilled practitioner would contemplate other nonpolar solvents whichcould be used herein.

“Non-polar light phase” indicates a non-polar solvent with a densityless than the corresponding aqueous phase.

“Solid binding material” indicates a solid substance that is able tobind one or more compounds of interest under the reaction orpurification conditions of interest. For example, a solid bindingmaterial may be used to bind one or more waxy impurities, and the waxyimpurities removed from the desired material by removal of the solidbinding material. In certain embodiments, including any of the foregoingembodiments, the solid binding material is a finely divided solid. Incertain embodiments, including any of the foregoing embodiments, thefinely divided solid comprises particles having an average size of about60 microns or less. In certain embodiments, including any of theforegoing embodiments, the solid binding material comprises carbon,silica, alumina, or diatomaceous earth. In some embodiments, the solidbinding material comprises finely divided carbon, silica, alumina, ordiatomaceous earth.

“Aqueous work up” indicates one or more of quenching a reaction todeactivate any unreacted reagents using, in some embodiments, an aqueoussolution; cooling the reaction mixture or adding an antisolvent(including an aqueous solution) to induce precipitation, and collectingor removing the solids by filtration, decantation, or centrifugation;and separating the reaction mixture into organic and aqueous layers byliquid-liquid extraction.

“Inorganic salt base” refers to a salt of a base, wherein the counterionis inorganic. In certain embodiments, including any of the foregoingembodiments, the inorganic salt base is sodium methoxide (NaOMe),lithium methoxide (LiOMe), potassium methoxide (KOMe), or magnesiummethoxide (Mg(OMe)₂). In certain embodiments, including any of theforegoing embodiments, the inorganic salt base is NaOMe.

The term “simulated moving bed apparatus” or “SMB apparatus” is usedherein to denote a continuous or semi continuous chromatographic processcomposed of a combination of columns, inlet and outlet valve manifolds,sources of feed stream and of eluent, and mechanism for the timedswitching of flows through the inlet and outlet manifolds, by way ofwhich simulated moving bed chromatography is practiced. A typical SMBsystem comprises a plurality of columns serially connected, each columnhaving an inlet manifold arranged to direct incoming flow from aplurality of inlet lines to the column and an outlet manifold arrangedto direct flow emerging from the column to a plurality of dischargelines; a source of a feed stream (also referred to as a “feed solution”)comprising the species to be separated; an eluent source; and acontroller for inlet and outlet valve manifolds that forms flow pathscommunicating each column with a selected inlet line and a selecteddischarge line, and for changing the inlet and discharge lines soselected at preselected time intervals.

A single manifold can serve as both the outlet manifold of one columnand the inlet manifold of another. In certain embodiments, including anyof the foregoing embodiments, the inlet manifold associated with eachcolumn joins the column with at least three, and preferably only three,inlet lines, while the outlet manifold associated with each columnlikewise joins the column with at least three, and preferably onlythree, discharge lines. Selections among the various lines in eachmanifold are achieved by conventional means, such as a remotelycontrolled on-off valve on each line or a remotely controlled multi-wayvalve. In certain embodiments, including any of the foregoingembodiments, feeds and withdrawals are performed simultaneously asfollows: (i) eluent is fed at an inlet manifold at the upstream end,(ii) extract is withdrawn at an outlet manifold between the upstream endand the downstream end, (iii) the feed stream of species to be separatedis fed through an inlet manifold between the extract withdrawal site andthe downstream end, and (iv) raffinate is withdrawn through an outletmanifold at the downstream end.

After a selected time interval (referred to as the “switch time” of thesystem), the valve positions in the inlet and outlet manifolds arereconfigured to advance feed and withdrawal sites, in a common directionaround the circuit, which is the same direction as the eluent flow. Thenew configuration is maintained for another selected time interval,which can either be of the same duration as the first or of a differentduration, and the various sites of feeding and withdrawal, are advancedagain. These incremental advancements are continued as feed solutioncontinues to be supplied to the system, and the result is a simulatedcontinuous-flow system. The pattern of introduction and withdrawal thusrotates around the column circuit.

The terms “extract” and “raffinate” are used herein as they are inconventional SMB terminology. Thus, “extract” denotes a fraction of theinitial liquid mixture that contains the component(s) that is/are morestrongly retained on the solid phase relative to the other component(s)and that elute(s) in a relatively purified form, i.e., relativelyisolated from the less strongly retained component(s). The term“raffinate” denotes a fraction of the initial liquid mixture thatcontains the components(s) that is/are retained relatively weakly on thesolid phase and that elute(s) in a relatively purified form, i.e.,relatively isolated from the strongly retained component(s).

The expression “serially connected in a circuit” in reference to thevarious columns in the SMB apparatus denotes that the columns are joinedby fluid transfer conduits through inlet and outlet manifolds that canbe arranged to cause the discharge from each column to be fed to anadjacent column in the series in a loop.

Variations that have been developed in the prior SMB processes of theprior art can be applied in an analogous manner to the process of thepresent invention. Thus, in the various changes of functions of themultifunctional ports, the isolation points separating the two groups ofcolumns and all introduction and withdrawal sites can be advanced eithersimultaneously or in an asynchronous or staged manner (as in the Varicolvariation, U.S. Pat. Nos. 6,136,198, 6,375,839, 6,413,419, and6,712,973) or at variable flow rates (as in the PowerFeed variation,U.S. Pat. No. 5,102,553). Simultaneous advancement is preferred. Incertain embodiments, including any of the foregoing embodiments, columnscan be operated in parallel groups as described in U.S. Pat. No.7,618,539 B2. Other useful operating strategies include Modicon (U.S.Pat. No. 7,479,228), SMB internal recirculation (U.S. Pat. No.8,282,831)

Operating parameters of the SMB apparatus, such as time intervalsbetween advancements of the various introduction and withdrawal sites,the lengths and widths of individual columns, pump pressures, and themass or volumetric flow rate through each column, will generally bewithin the ranges used in SMB systems known in the art. Typical columnsare packed-bed columns with lengths ranging from 5 to 15 cm anddiameters ranging from 2 mm to 1,600 mm. Volumetric relative flow ratescalculated per unit column cross section will generally be between 0.5mL/min/cm² and 40 mL/min/cm²; pump pressures will generally be between 2bar and 60 bar, and switch times will generally be from about 0.15minutes to about 15 minutes.

“Area percent” refers to a percent area under the peak for a compound inan ultra-high performance liquid chromatography (UPLC) or highperformance liquid chromatography (HPLC) sample as measured at adesignated wavelength. In certain embodiments, including any of theforegoing embodiments, the area percent is measured as a UPLC responseat 210 nm compared to the total area of all peaks in the chromatogram.Area percent can be denoted by “A %” or “(A) %” in the methods disclosedherein.

“Weight percent” is based on the total weight of the material obtainedand refers to a percent weight of a compound as measured in a UPLCresponse at a designated wavelength compared to a UPLC response of astandard. In certain embodiments, including any of the foregoingembodiments, the weight percent is measured as a UPLC response at 210 nmcompared to a pure tocotrienol standard. In certain embodiments,including any of the foregoing embodiments, the weight percent ismeasured as a UPLC response at 210 nm compared to a purealpha-tocotrienol standard. Weight percent can be denoted by “wt %” or“wt. %” or “% (w/w)” in the methods disclosed herein.

The terms “early-eluting impurity compound(s)” and “early-elutingimpurities” include any compounds eluting earlier than alphatocotrienol. In certain embodiments, including any of the foregoingembodiments, the early-eluting impurity compound is anon-alpha-tocotrienol, i.e., the early-eluting impurity is a tocotrienolwhich is not alpha-tocotrienol.

The terms “late-eluting impurity compound(s)” and “late-elutingimpurities” include any compounds eluting later than alpha tocotrienol.In certain embodiments, including any of the foregoing embodiments, thelate-eluting impurity compound is one or more tocopherols, or analpha-tocotrienol-MeOH adduct, where “MeOH” denotes methanol. In certainembodiments, including any of the foregoing embodiments, thelate-eluting impurity compound is α-tocopherol.

The term “substantially free of” or “substantially in the absence of”with respect to a composition refers to a composition that includes atleast 85% or 90% by weight, in certain embodiments at least 95%, 98%,99% or 100% by weight, of a designated enantiomer or stereoisomer of acompound. In certain embodiments, including any of the foregoingembodiments, in the methods and compounds provided herein, the compoundsare substantially free of other enantiomers or stereoisomers.

Similarly, the term “isolated” with respect to a composition refers to acomposition that includes at least 85%, 90%, 95%, 98%, 99% to 100% byweight, of a designated compound, enantiomer, or stereoisomer, theremainder comprising other chemical species, enantiomers, orstereoisomers.

Methods

Enriched alpha-tocotrienol (AT3) compositions can be obtained from tocolmixtures by the methods of the present invention. In particular, highlevels of alpha-tocotrienol may be obtained by the methods of thepresentation invention. The methods disclosed herein can advantageouslybe used to obtain high levels of alpha-tocotrienol from tocol mixturesthat contain relatively high amounts of non-tocol compounds. Forexample, the methods disclosed herein can be applied to tocol mixtureshaving at least 5% phytosterol content. In addition, the methodsdisclosed herein allow improved recovery of alpha-tocotrienol initiallypresent in the tocol mixtures due to fewer or no separation stepsbetween step (a) and step (b) as described below. Moreover, the methodsdisclosed herein produce alpha-tocotrienol of high purity using asimulated moving bed (SMB) process.

In one aspect of the invention, a method for making an alpha-tocotrienolenriched tocol mixture is provided, comprising: (a) contacting a tocolmixture with an amino-alkylating agent, wherein the tocol mixturecomprises at least one non-alpha tocotrienol, at least one non-tocol,optionally alpha tocotrienol, and optionally one or more tocopherols,whereby the non-alpha tocotrienols are amino-alkylated; (b) reducing theamino-alkylated non-alpha tocotrienols to alpha tocotrienol with areducing agent; (c) removing one or more waxy impurities from themixture; (d) contacting the mixture with an agent that binds one or morepolar impurities; and (e) removing the agent. In some embodiments,including any of the foregoing embodiments, the tocol mixture is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% tocols. In someembodiments, including any of the foregoing embodiments, there is nointervening step to separate components of the mixture between step (a)and step (b). In some embodiments, including any of the foregoingembodiments, there is no intervening step to separate components of themixture between step (a) and step (b) other than an optional step toremove water formed from the reaction of step (a).

In some embodiments, the method further includes steps (c)(1) to (c)(4)after step (c), wherein: step (c)(1) comprises converting the alphatocotrienol to an alpha tocotrienol salt; step (c)(2) comprisesproviding a polar solvent phase and a non-polar solvent phase andpartitioning the alpha tocotrienol salt to the polar solvent phase; step(c)(3) comprises removing the non-polar solvent phase; and step (c)(4)comprises converting the alpha tocotrienol salt in the polar solventphase to alpha tocotrienol.

In some embodiments, including any of the foregoing embodiments, themethod further includes a step (a)(1) after step (a), wherein step(a)(1) comprises removing water formed from the reaction of step (a).

In another aspect of the invention, a method for making analpha-tocotrienol enriched tocol mixture is provided, comprising: (a)contacting a tocol mixture with an amino-alkylating agent, wherein thetocol mixture comprises at least one non-alpha tocotrienol, at least onenon-tocol, optionally alpha tocotrienol, and optionally one or moretocopherols, whereby the non-alpha tocotrienols are amino-alkylated; (b)reducing the amino-alkylated non-alpha tocotrienols to alpha tocotrienolwith a reducing agent; (c) removing one or more waxy impurities from themixture; (c)(1) converting the alpha tocotrienol to an alpha tocotrienolsalt; (c)(2) providing a polar solvent phase and a non-polar solventphase and partitioning the alpha tocotrienol salt to the polar solventphase; (c)(3) removing the non-polar solvent phase; (c)(4) convertingthe alpha tocotrienol salt from in polar solvent phase to alphatocotrienol; (d) contacting the mixture with an agent that binds one ormore polar impurities; and (e) removing the agent. In some embodiments,including any of the foregoing embodiments, there is no step to separatecomponents of the mixture between step (a) and step (b). In someembodiments, including any of the foregoing embodiments, the methodfurther includes a step (a)(1) after step (a), wherein step (a)(1)comprises removing water formed from the reaction of step (a). In someembodiments, including any of the foregoing embodiments, the tocolmixture is at least about 50%, 55%, 60%, 65%, or 70% tocols.

In some embodiments, step (a)(1) comprises any method known in the artfor removing water from a mixture, which is formed as a by-product ofthe amino-alkylation reaction. In some embodiments, including any of theforegoing embodiments, step (a)(1) comprises contacting the mixture witha drying agent. Suitable drying agents include, but are not limited to,for example, magnesium sulfate or sodium sulfate. In some embodiments,including any of the foregoing embodiments, step (a)(1) comprises anaqueous wash with brine, followed by contacting the mixture with adrying agent. In some embodiments, including any of the foregoingembodiments, step (a)(1) is carried out in the presence of an organicsolvent such as, for example, toluene.

In some embodiments, including any of the foregoing embodiments, step(a)(1) comprises an azeotropic distillation to remove the water. In someembodiments, including any of the foregoing embodiments, step (a)(1)comprises azeotropic distillation in the presence of a solvent with aboiling point of at least 80° C. at 1 atm pressure. In some embodiments,including any of the foregoing embodiments, step (a)(1) comprisesazeotropic distillation in the presence of a solvent with a boilingpoint of at least 95° C. at 1 atm pressure. Suitable solvents includethose solvents that can form an azeotrope with water, including, e.g.,branched alcohols with a boiling point higher than 80° C. Exemplarysolvents include, but are not limited to, 2-butanol, 2-pentanol,2-methyl-2-butanol, ethanol, toluene, ethyl acetate, acetonitrile,methyl ethyl ketone, cyclohexanol, 2-pentanol, 2-hexanol, or2-methyl-1-propanol. In some embodiments, the solvent is2-methyl-2-butanol (also referred to as “t-amyl alcohol” or “t-AmOH”).

In some embodiments, including any of the foregoing embodiments, theamino-alkylating agent in step (a) comprises a secondary amine and aformaldehyde equivalent. In some embodiments, including any of theforegoing embodiments, the secondary amine is a cyclic amine (e.g.,morpholine, piperidine, pyrrolidine, and N-methyl-piperazine) or adialkylamines (e.g., dimethylamine, diethylamine, diisopropylamine). Insome embodiments, including any of the foregoing embodiments, thesecondary amine is N-methyl-piperazine. In some embodiments, includingany of the foregoing embodiments, about 2 to about 5 equivalents of thesecondary amine with respect to the tocol mixture is used; in someembodiments, including any of the foregoing embodiments, about 2equivalents, about 2.5 equivalents, about 3 equivalents, about 3.5equivalents, about 4 equivalents, about 4.5 equivalents, or about 5equivalents is used. In some embodiments, including any of the foregoingembodiments, the formaldehyde equivalent is paraformaldehyde, a formalinsolution (30-40% formaldehyde in water), 1,3,5-trioxane, formaline,formaldehyde gas, or hexamethylenetetramine. In some embodiments,including any of the foregoing embodiments, the formaldehyde equivalentis paraformaldehyde. In some embodiments, including any of the foregoingembodiments, about 1.25 to about 3.5 equivalents of the formaldehydeequivalent with respect to the tocol mixture is used; in someembodiments, including any of the foregoing embodiments, about 1.25equivalents, about 1.5 equivalents, about 1.75 equivalents, about 2equivalents, about 2.25 equivalents, about 2.5 equivalents, about 2.75equivalents, about 3 equivalents, or about 3.5 equivalents is used. Thetemperature of step (a) can be any temperature deemed suitable by theskilled practitioner. In some embodiments, including any of theforegoing embodiments, the temperature in step (a) before the tocolmixture is contacted with the amino-alkylating agent is about 70° C., orabout 75° C., or about 80° C. In some embodiments, including any of theforegoing embodiments, the temperature in step (a) after the tocolmixture is contacted with the amino-alkylating agent is about 100° C.,or about 105° C., or about 110° C. In some or any embodiments of step(a) after the amino-alkylated product is formed, the temperature islowered by the addition of a solvent, such as 2-methyl-2-butanol.

In some embodiments, including any of the foregoing embodiments, thereducing agent in step (b) is a borohydride. The borohydride can be; forexample, sodium cyano borohydride (also referred to as “NaCNBH₃” or“NaBH₃CN”), sodium borohydride, lithium borohydride, or zincborohydride. In some embodiments, including any of the foregoingembodiments, the reducing agent in step (b) is NaBH₃CN. In someembodiments, including any of the foregoing embodiments, about 4 toabout 6 equivalents of the reducing agent with respect to the tocolmixture is used; in some embodiments; including any of the foregoingembodiments, about 4 equivalents, about 4.5 equivalents, about 4.75equivalents, about 5 equivalents, about 5.25 equivalents, about 5.5equivalents, or about 6 equivalents is used. In some embodiments,including any of the foregoing embodiments, an alcohol having a boilingpoint of at least about 95° C. is used as a solvent in step (b). In someembodiments, including any of the foregoing embodiments, the solvent instep (b) is a four or five carbon alcohol. In some embodiments,including any of the foregoing embodiments, the solvent in step (b) is2-hydroxybutanol, 2-hydroxypentanol, or 2-methyl-2-butanol. In someembodiments, including any of the foregoing embodiments, the solvent instep (b) is 2-methyl-2-butanol. Tn some embodiments, including any ofthe foregoing embodiments, the temperature in step (b) is about 70° C.,or about 75° C., or about 80° C. In some embodiments, including any ofthe foregoing embodiments, the temperature in step (b) is raised toabout 100° C., or about 105° C., or about 107° C., or about 110° C. Insome embodiments, including any of the foregoing embodiments, theprogression of step (b) can be monitored by HPLC by measuring the amountof starting material remaining. In some embodiments, including any ofthe foregoing embodiments, step (b) comprises a water quench subsequentto addition of solvent, wherein the water quench and resulting phasesplit of the reduction step is performed at 60° C. to minimize saltprecipitation. In some embodiments; including any of the foregoingembodiments, step (b) comprises a water quench followed by addition ofsolvent (e.g., heptane or n-heptane), wherein the water quench andresulting phase split of the reduction step is performed at about 20°C.-25° C., after which insoluble solids are filtered.

In some embodiments, including any of the foregoing embodiments, one ormore by-products are produced in steps (a) and/or (b), and wherein themixture optionally comprises residual secondary amine, and wherein themethod comprises a step (b)(1) after step (b), wherein step (b)(1)comprises removing at least one of the one or more by-products producedin steps (a) and/or (b) and the optional residual secondary amine. Insome embodiments, including any of the foregoing embodiments, step(b)(1) comprises an aqueous work up. In some embodiments, including anyof the foregoing embodiments, step (b)(1) comprises contacting themixture with a solvent and water, filtering the mixture to removeinsoluble solid byproducts, and an aqueous work-up of the filtrate. Insome embodiments, including any of the foregoing embodiments, theaqueous work up comprises: (i) contacting the mixture with isopropylacetate, water, and Na₂HPO₄, whereby an aqueous phase is formed; (ii)removing the aqueous phase; (iii) performing an acid wash on thenon-aqueous phase; and iv) removing the aqueous phase. In someembodiments, the aqueous work up comprises: (i) contacting the mixturewith isopropyl acetate and water, whereby an aqueous phase is formed;(ii) removing the aqueous phase; (iii) performing an acid wash on thenon-aqueous phase; and iv) removing the aqueous phase. In someembodiments, including any of the foregoing embodiments, the aqueouswork up comprises: (i) contacting the mixture with isopropyl acetate andwater, whereby an aqueous phase is formed; (ii) removing the aqueousphase; (iii) contacting the mixture with water, whereby an aqueous phaseis formed, and removing the aqueous phase; and (iv) performing an acidwash on the non-aqueous phase; and v) removing the aqueous phase. Insome embodiments, including any of the foregoing embodiments, the acidwash comprises citric acid. In some embodiments, the acid wash comprisesabout 5% (w/w), about 7% (w/w), about 10% (w/w), about 12% (w/w), about15% (w/w), about 18% (w/w), or about 20 (w/w) citric acid. In someembodiments, including any of the foregoing embodiments, the aqueouswork up is conducted at about 55° C., at about 60° C., or at about 65°C.

In some embodiments, including any of the foregoing embodiments, step(c) comprises removing solvent that may be present in the mixture and/orcooling to precipitate the one or more waxy impurities. In someembodiments, including any of the foregoing embodiments, some of thesolvent which may be present is removed by distillation. In someembodiments, including any of the foregoing embodiments, step (c)comprises performing a solvent exchange to a polar protic solvent. Insome embodiments, including any of the foregoing embodiments, the polarprotic solvent is methanol. In some embodiments, including any of theforegoing embodiments, step (c) comprises reducing the mixture to abouta minimum stir volume. In some embodiments, including any of theforegoing embodiments, step (c) comprises (i) performing a solventexchange to methanol, (ii) reducing the mixture to about a minimum stirvolume, and (iii) cooling the mixture. In some embodiments, includingany of the foregoing embodiments, the cooling in step (c) comprisescooling to about −35° C. to about 5° C. In some embodiments, includingany of the foregoing embodiments, the cooling in step (c) comprisescooling to about −20° C. to about −10° C. In some embodiments, includingany of the foregoing embodiments, the cooling in step (c) comprisescooling to about −35° C. to about −15° C. In some embodiments, includingany of the foregoing embodiments, removing the one or more waxyimpurities comprises contacting the mixture with a solid bindingmaterial. In some embodiments, including any of the foregoingembodiments, removing the one or more waxy impurities comprisescontacting the mixture with the solid binding material to produce aslurry, followed by filtering the slurry and retaining the filtrate. Insome embodiments, including any of the foregoing embodiments, the solidbinding material is a finely divided solid comprising carbon, silica,alumina, or diatomaceous earth. In some embodiments, including any ofthe foregoing embodiments, the solid binding material is Celite®(diatomaceous earth).

In some embodiments, including any of the foregoing embodiments, step(c) comprises holding the mixture at a temperature of about 15° C. toabout 35° C. for a duration of from about 1 to 4 hours and filtering themixture to remove insoluble byproducts. In some embodiments, the mixtureis held at a temperature of from about 15° C. to about 25° C. In someembodiments, the mixture is held at a temperature of from about 20° C.to about 30° C. In some embodiments, the mixture is held at atemperature of from about 22° C. to about 28° C. In some embodiments,the mixture is held at a temperature of about 15° C., 20° C., 25° C.,30° C., or about 35° C. In some embodiments, including any of theforegoing embodiments, the mixture is held at a set temperature ortemperature range for from about 1 to about 3 hours, or from about 1 toabout 2 hours, or from about 2 to about 4 hours. In some embodiments,including any of the foregoing embodiments, the mixture is held at a settemperature or temperature range for about 1 hour, 2 hours, 3 hours, or4 hours. In some embodiments, including any of the foregoingembodiments, step (c) comprises an aqueous work up of the mixture. Insome embodiments, including any of the foregoing embodiments, theaqueous work up comprises: (i) contacting the mixture with water,whereby an aqueous phase is formed; (ii) removing the aqueous phase;(iii) performing an acid wash on the non-aqueous phase; (iv) removingthe aqueous phase. In some embodiments, including any of the foregoingembodiments, the aqueous workup further comprises (v) performing a washon the non-aqueous phase with water and/or aqueous sodium bicarbonate(NaHCO₃) and subsequently removing the aqueous phase. In someembodiments, including any of the foregoing embodiments, the acid washcomprises citric acid. In some embodiments, the acid wash comprisesabout 5% (w/w), about 7% (w/w), about 10% (w/w), about 12% (w/w), about15% (w/w), about 18% (w/w), or about 20 (w/w) citric acid.

In some embodiments, including any of the foregoing embodiments, asecond solvent is optionally added before the alpha tocotrienol isconverted to a salt in step (c)(1). In some embodiments, including anyof the foregoing embodiments, the second solvent is a polar solvent suchas N-methyl-pyrrolidone (NMP). In some embodiments, including any of theforegoing embodiments, the alpha tocotrienol in step (c)(1) is convertedto the alpha tocotrienol salt with an inorganic salt base having apK_(a) of more than about 10. In some embodiments, including any of theforegoing embodiments, the inorganic salt base is selected from thegroup consisting of sodium methoxide (NaOMe), lithium methoxide (LiOMe),potassium methoxide (KOMe), or magnesium methoxide (Mg(OMe)₂). In someembodiments, including any of the foregoing embodiments, the inorganicsalt base is NaOMe.

In some embodiments, including any of the foregoing embodiments, thepolar solvent phase in step (c)(2) comprises NMP and/or methanol (MeOH).In some embodiments, including any of the foregoing embodiments, thepolar solvent phase in step (c)(2) comprises NMP and MeOH. In someembodiments, including any of the foregoing embodiments, the non-polarsolvent phase in step (c)(2) comprises an alkane. In some embodiments,including any of the foregoing embodiments, the non-polar solvent phasein step (c)(2) comprises a hexane or a heptane. In some embodiments,including any of the foregoing embodiments, the non-polar solvent phasein step (c)(2) comprises n-heptane. In some embodiments, including anyof the foregoing embodiments, the partitioning between the polar andnon-polar solvent phases is performed at less than 10° C. In someembodiments, including any of the foregoing embodiments, thepartitioning between the polar and non-polar solvent phases is performedat about 0° C.

In some embodiments, including any of the foregoing embodiments,converting the alpha tocotrienol salt to alpha tocotrienol in step(c)(4) comprises addition of an acid having a pH of less than about 7.In some embodiments, including any of the foregoing embodiments,converting the alpha tocotrienol salt to alpha tocotrienol in step(c)(4) comprises addition of HCl; alternatively, in some embodiments,including any of the foregoing embodiments, converting the alphatocotrienol salt to alpha tocotrienol in step (c)(4) comprises additionof a water-soluble acid with a pKa of 7 or less, for example: aceticacid, formic acid, HBr, H₃PO₄, H₂SO₄, and carbonic acid. In someembodiments, including any of the foregoing embodiments, step (c)(4)comprises production of a salt by-product, and wherein the methodcomprises a step (c)(5) after step (c)(4), wherein step (c)(5) comprisesremoving the salt by-product produced in step (c)(4). In someembodiments, including any of the foregoing embodiments, step (c)(5)comprises solvent extraction with a non-polar light phase, followed byretaining the non-polar light phase. In some embodiments, including anyof the foregoing embodiments, the non-polar light phase comprisestoluene, any isomers of hexane, heptane, pentane, xylene, or petroleumether. In some embodiments, including any of the foregoing embodiments,the non-polar light phase is washed with an aqueous wash. In someembodiments, including any of the foregoing embodiments, the non-polarlight phase is washed with a brine solution.

In some embodiments, including any of the foregoing embodiments, theagent in step (d) comprises silica, alumina, diatomaceous earth, orclays. In some embodiments, including any of the foregoing embodiments,the solvent(s) in the nonpolar light phase are removed, such as bydistillation.

In some embodiments, steps (d) and (e) comprise adding the mixture to acolumn comprising the agent, and eluting the mixture off the column. Insome or any embodiments, solvent is removed to prepare a feed stream ata concentration suitable for SMB purification.

In some embodiments, including any of the foregoing embodiments, themethod comprises a step (f) after step (e), wherein step (f) comprisesperforming a solvent exchange of the mixture with a solvent that ismethanol or acetonitrile. In some embodiments, including any of theforegoing embodiments, the solvent exchange is with methanol. In someembodiments, including any of the foregoing embodiments, the solventexchange is with acetonitrile. In some embodiments, including any of theforegoing embodiments, the solvent exchange is performed in the absenceof light and/or protected from exposure to sunlight.

In some embodiments, including any of the foregoing embodiments, themethod is performed in the absence of light and/or protected fromexposure to sunlight. In some embodiments, including any of theforegoing embodiments, the method partially performed in the absence oflight and/or protected from exposure to sunlight. For example, one ormore of any of steps (a), (a)(1), (b), (b)(1), (c), (c)(1), (c)(2),(c)(3), (c)(4), (c)(5), (e) and (f) as disclosed herein can be performedin the absence of light and/or protected from exposure to sunlight.

In some or any embodiments, solvent is removed from the productcomposition to prepare a feed stream at a concentration suitable for SMBpurification.

In some embodiments, including any of the foregoing embodiments, thetocol mixture in step (a) is a plant, plant extract, or plant-derivedmaterial. In some embodiments, including any of the foregoingembodiments, the tocol mixture is a palm oil or a material derived frompalm oil, a palm fruit extract, or a mixture of palm oil and palm fruitextract. In some embodiments, including any of the foregoingembodiments, the tocol mixture is a palm oil. In some embodiments,including any of the foregoing embodiments, the tocol mixture is derivedfrom a palm oil. In some embodiments, the tocol mixture is acommercially available product comprising an enriched tocotrienolextract derived from palm oil. For example, the commercially availableproduct can be one or more products as provided by Carotech, Golden HopeBioorganic, Davos Life Science, Beijing Gingko Group, Eisai, EastmanCorporation, Oryza Oil & Fat Company, Sime Darby Biorganic Sdn Bhd orPalm Nutraceuticals. In some embodiments, the tocol mixture is Tocomin50®. In some embodiments, including any of the foregoing embodiments,the phytosterol content of the tocol mixture is at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%,at least about 10%, at least about 11%, at least about 12%, or at leastabout 13%, wherein percentage is provided as a weight or area percent.In some embodiments, including any of the foregoing embodiments, thetocol mixture comprises at least about 25 wt. % tocols. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 30 wt % tocols. In some embodiments,including any of the foregoing embodiments, the tocol mixture comprisesat least about 35 wt. % tocols. In some embodiments, including any ofthe foregoing embodiments, the tocol mixture comprises at least about 40wt. % tocols. In some embodiments, including any of the foregoingembodiments, the tocol mixture comprises at least about 45 wt. % tocols.In some embodiments, including any of the foregoing embodiments, thetocol mixture comprises at least about 50 wt. % tocols. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 55 wt. % tocols. In some embodiments,including any of the foregoing embodiments, the tocol mixture comprisesat least about 60 wt. % tocols. In some embodiments, including any ofthe foregoing embodiments, the tocol mixture comprises 65 wt. % tocols.In some embodiments, including any of the foregoing embodiments, thetocol mixture comprises at least about 66 wt. % tocols. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 67 wt. % tocols. In some embodiments,including any of the foregoing embodiments, the tocol mixture comprisesat least about 68 wt. % tocols. In some embodiments, including any ofthe foregoing embodiments, the tocol mixture comprises at least about 69wt. % tocols. In some embodiments, including any of the foregoingembodiments, the tocol mixture comprises at least about 70 wt. % tocols.In some embodiments, the tocol mixture is Gold Tri.E™ 70. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 75 wt. % tocols. In some embodiments,including any of the foregoing embodiments, the tocol mixture comprisesat least about 76 wt. % tocols. In some embodiments, including any ofthe foregoing embodiments, the tocol mixture comprises at least about 77wt. % tocols. In some embodiments, including any of the foregoingembodiments, the tocol mixture comprises at least about 78 wt. % tocols.In some embodiments, including any of the foregoing embodiments, thetocol mixture comprises at least about 79 wt. % tocols. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 80 wt. % tocols. In some embodiments,including any of the foregoing embodiments, the tocol mixture comprisesat least about 81 wt. % tocols. In some embodiments, including any ofthe foregoing embodiments, the tocol mixture comprises at least about 82wt. % tocols. In some embodiments, including any of the foregoingembodiments, the tocol mixture comprises at least about 83 wt. % tocols.In some embodiments, including any of the foregoing embodiments, thetocol mixture comprises at least about 84 wt. % tocols. In someembodiments, including any of the foregoing embodiments, the tocolmixture comprises at least about 85 wt. % tocols. In some embodiments,the tocol mixture is natural palm oil that is a high purity palmtocotrienol product from e.g., Davos Life Science. In some embodiments,the tocol mixture in step (a) contains at least about 65 (A) % tocols,at least about 68 (A) % tocols, or at least about 70 (A) % tocols. Insome embodiments, the tocol mixture in step (a) contains about about 84(A) % of a mixture of tocotrienols and tocopherols.

In some embodiments, including any of the foregoing embodiments, thealpha-tocotrienol in the product composition is the natural form ofalpha-tocotrienol, i.e.:

In various embodiments, including any of the foregoing embodiments, thealpha-tocotrienol in the product composition is at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% the natural form of alpha-tocotrienol.

In some embodiments, including any of the foregoing embodiments, theproduct composition produced by the method for making analpha-tocotrienol enriched tocol mixture comprises at least about 40 wt.%, at least about 41 wt. %, at least about 42 wt. %, at least about 43wt. %, at least about 44 wt. %, at least about 45 wt. %, at least about46 wt. %, at least about 47 wt. %, at least about 48 wt. %, at leastabout 49 wt. %, at least about 50 wt. %, at least about 51 wt. %, atleast about 52 wt. %, at least about 53 wt. %, at least about 54 wt. %,at least about 55 wt. %, at least about 56 wt. %, at least about 57 wt.%, or at least about 58 wt. % alpha-tocotrienol. In some embodiments,including any of the foregoing embodiments, the product compositioncomprises at least about 59 wt. %, at least about 60 wt. %, at leastabout 61 wt. %, at least about 62 wt. %, at least about 63 wt. %, atleast about 64 wt. %, at least about 65 wt. %, at least about 66 wt. %,at least about 67 wt. %, at least about 68 wt. %, at least about 69 wt.%, or at least about 70 wt. % alpha-tocotrienol.

In some embodiments, including any of the foregoing embodiments, theproduct composition produced by the method for making analpha-tocotrienol enriched tocol mixture comprises at least about 50 (A)%, at least about 51 (A) %, at least about 52 (A) %, at least about 53(A) %, at least about 54 (A) %, at least about 55 (A) %, at least about56 (A) %, at least about 57 (A) %, or at least about 58 (A) % weightpercent alpha-tocotrienol as determined by HPLC or UPLC. In someembodiments, including any of the foregoing embodiments, the productcomposition comprises at least about 59 (A) %, at least about 60 (A) %,at least about 61 (A) %, at least about 62 (A) %, at least about 63 (A)%, at least about 64 (A) %, at least about 65 (A) %, at least about 66(A) %, at least about 67 (A) %, at least about 68 (A) %, at least about69 (A) %, or at least about 70 (A) % alpha-tocotrienol as determined byHPLC or UPLC.

In some embodiments, including any of the foregoing embodiments, theproduct composition produced by the method for making analpha-tocotrienol enriched tocol mixture comprises at least about 60%,at least about 61%, at least about 62%, at least about 63%, at leastabout 64%, at least about 65%, at least about 66%, at least about 67%,at least about 68%, at least about 69%, at least about 70%, at leastabout 71%, at least about 72%, at least about 73%, at least about 74%,at least about 75%, at least about 76%, at least about 77%, at leastabout 78%, at least about 79%, at least about 80%, at least about 81%,at least about 82%, at least about 83%, at least about 84%, or at leastabout 85% of the mass of the starting amount of tocol mixture used instep (a).

Also provided herein are methods of purifying a tocotrienol by simulatedmoving bed chromatography. The methods begin with a feed stream (alsoreferred to as a “feed solution”) comprising the tocotrienol and providea raffinate comprising the tocotrienol in purified form. In certainembodiments, including any of the foregoing embodiments, the methodsbegin with a feed stream comprising alpha-tocotrienol and provide araffinate comprising alpha-tocotrienol in purified form. The raffinateis purified relative to the feed stream. In certain embodiments,including any of the foregoing embodiments, the raffinate issubstantially purified relative to the feed stream.

In some embodiments, including any of the foregoing embodiments, themethods of purifying a tocotrienol as disclosed herein yield tocotrienolof high purity. In some embodiments, including any of the foregoingembodiments, the tocotrienol purity is in the range of from about 80 wt.% to 99.9 wt. %, or in the range of from about 85 wt. % to 99.9 wt. %,or in the range of from about 90 wt. % to 99.9 wt. %, or in the range offrom about 95 wt. % to 99.9 wt. %. In some embodiments, including any ofthe foregoing embodiments, the tocotrienol purity is in the range offrom about 80 wt. % to about 99.9 wt. %, or in the range of from about85 wt. % to about 99.9 wt. %, or in the range of from about 90 wt. % toabout 99.9 wt. %, or in the range of from about 95 wt. % to about 99.9wt. %. In some embodiments, including any of the foregoing embodiments,the tocotrienol purity is greater than 80 wt. %, or greater than 85 wt.%, or greater than 90 wt. %, or greater than 91 wt. %, or greater than92 wt. %, or greater than 93 wt. %, or greater than 94 wt. %, or greaterthan 95 wt. %, or greater than 96 wt. %, or greater than 97 wt. %, orgreater than 98 wt. %, or greater than 99 wt. %, or greater than 99.5wt. %, or greater than 99.9 wt. %. In some embodiments, including any ofthe foregoing embodiments, the tocotrienol purity is more than about 80wt. %, or more than about 85 wt. %, or more than about 90 wt. %, or morethan about 91 wt. %, or more than about 92 wt. %, or more than about 93wt. %, or more than about 94 wt. %, or more than about 95 wt. %, or morethan about 96 wt. %, or more than about 97 wt. %, or more than about 98wt. %, or more than about 99 wt. %, or more than about 99.5 wt. %, ormore than about 99.9 wt. %. In some embodiments, including any of theforegoing embodiments, the impurities in the final product are less thanabout 20 wt. %, or less than about 15 wt. %, or less than about 10 wt.%, or less than about 5 wt. %, or less than about 4 wt. %, or less thanabout 3 wt. %, or less than about 2 wt. %, or less than about 1 wt. %,or less than about 0.5 wt. %, or less than about 0.1 wt. %. In someembodiments, including any of the foregoing embodiments, the impuritiesin the final product comprise tocols or tocol derivatives in the finalproduct and total less than about 5 wt. %, less than about 4 wt. %, lessthan about 3 wt. %, less than about 2 wt. %, less than about 1 wt. %,less than about 0.5 wt. % or less than about 0.1 wt. %. In someembodiments, including any of the foregoing embodiments, the impuritiesin the final product consist of tocols or tocol derivatives in the finalproduct and total less than about 5 wt. %, less than about 4 wt. %, lessthan about 3 wt. %, less than about 2 wt. %, less than about 1 wt. %,less than about 0.5 wt. % or less than about 0.1 wt. %. In someembodiments, solvents which can be readily removed by evaporation arenot considered as impurities when determining the percentage purity of acomposition or the percentage of impurities present in a composition.

In some embodiments, including any of the foregoing embodiments, thetocotrienol purity is in the range of from about 80 (A) % to 99.9 (A) %,or in the range of from about 85 (A) % to 99.9 (A) %, or in the range offrom about 90 (A) % to 99.9 (A) %, or in the range of from about 95 (A)% to 99.9 (A) %. In some embodiments, including any of the foregoingembodiments, the tocotrienol purity is in the range of from about 80 (A)% to about 99.9 (A) %, or in the range of from about 85 (A) % to about99.9 (A) %, or in the range of from about 90 (A) % to about 99.9 (A) %,or in the range of from about 95 (A) % to about 99.9 (A) %. In someembodiments, including any of the foregoing embodiments, the tocotrienolpurity is greater than 80 (A) %, or greater than 85 (A) %, or greaterthan 90 (A) %, or greater than 91 (A) %, or greater than 92 (A) %, orgreater than 93 (A) %, or greater than 94 (A) %, or greater than 95 (A)%, or greater than 96 (A) %, or greater than 97 (A) %, or greater than98 (A) %, or greater than 99 (A) %, or greater than 99.5 (A) %, orgreater than 99.9 (A) %. In some embodiments, including any of theforegoing embodiments, the tocotrienol purity is more than about 80 (A)%, or more than about 85 (A) %, or more than about 90 (A) %, or morethan about 91 (A) %, or more than about 92 (A) %, or more than about 93(A) %, or more than about 94 (A) %, or more than about 95 (A) %, or morethan about 96 (A) %, or more than about 97 (A) %, or more than about 98(A) %, or more than about 99 (A) %, or more than about 99.5 (A) %, ormore than about 99.9 (A) %. In some embodiments, including any of theforegoing embodiments, the impurities in the final product are less thanabout 20 (A) %, or less than about 15 (A) %, or less than about 10 (A)%, or less than about 5 (A) %, or less than about 4 (A) %, or less thanabout 3 (A) %, or less than about 2 (A) %, or less than about 1 (A) %,or less than about 0.5 (A) %, or less than about 0.1 (A) %. In someembodiments, including any of the foregoing embodiments, the impuritiesin the final product comprise tocols or tocol derivatives in the finalproduct and total less than about 5 (A) %, less than about 4 (A) %, lessthan about 3 (A) %, less than about 2 (A) %, less than about 1 (A) %,less than about 0.5 (A) % or less than about 0.1 (A) %. In someembodiments, including any of the foregoing embodiments, the impuritiesin the final product consist of tocols or tocol derivatives in the finalproduct and total less than about 5 (A) %, less than about 4 (A) %, lessthan about 3 (A) %, less than about 2 (A) %, less than about 1 (A) %,less than about 0.5 (A) % or less than about 0.1 (A) %. In someembodiments, solvents which can be readily removed by evaporation arenot considered as impurities when determining the percentage purity of acomposition or the percentage of impurities present in a composition.

In some embodiments, including any of the foregoing embodiments, themethods of purifying a tocotrienol as disclosed herein yieldalpha-tocotrienol of high purity. In some embodiments, including any ofthe foregoing embodiments, the alpha-tocotrienol purity is in the rangeof from about 80 wt. % to 99.9 wt. %, or in the range of from about 85wt. % to 99.9 wt. %, or in the range of from about 90 wt. % to 99.9 wt.%, or in the range of from about 95 wt. % to 99.9 wt. %. In someembodiments, including any of the foregoing embodiments, thealpha-tocotrienol purity is in the range of from about 80 wt. % to about99.9 wt. %, or in the range of from about 85 wt. % to about 99.9 wt. %,or in the range of from about 90 wt. % to about 99.9 wt. %, or in therange of from about 95 wt. % to about 99.9 wt. %. In some embodiments,including any of the foregoing embodiments, the alpha-tocotrienol purityis greater than 80 wt. %, or greater than 85 wt. %, or greater than 90wt. %, or greater than 91 wt. %, or greater than 92 wt. %, or greaterthan 93 wt. %, or greater than 94 wt. %, or greater than 95 wt. %, orgreater than 96 wt. %, or greater than 97 wt. %, or greater than 98 wt.%, or greater than 99 wt. %, or greater than 99.5 wt. %, or greater than99.9 wt. %. In some embodiments, including any of the foregoingembodiments, the alpha-tocotrienol purity is more than about 80 wt. %,or more than about 85 wt. %, or more than about 90 wt. %, or more thanabout 91 wt. %, or more than about 92 wt. %, or more than about 93 wt.%, or more than about 94 wt. %, or more than about 95 wt. %, or morethan about 96 wt. %, or more than about 97 wt. %, or more than about 98wt. %, or more than about 99 wt. %, or more than about 99.5 wt. %, ormore than about 99.9 wt. %. In some embodiments, including any of theforegoing embodiments, the impurities in the final product are less thanabout 20 wt. %, or less than about 15 wt. %, or less than about 10 wt.%, or less than about 5 wt. %, or less than about 4 wt. %, or less thanabout 3 wt. %, or less than about 2 wt. %, or less than about 1 wt. %,or less than about 0.5 wt. %, or less than about 0.1 wt. %. In someembodiments, including any of the foregoing embodiments, the impuritiescomprise tocols or tocol derivatives in the final product and total lessthan about 5 wt. %, less than about 4 wt. %, less than about 3 wt. %,less than about 2 wt. %, less than about 1 wt. %, less than about 0.5wt. % or less than about 0.1 wt. %. In some embodiments, including anyof the foregoing embodiments, the impurities consist of tocols or tocolderivatives in the final product and total less than about 5 wt. %, lessthan about 4 wt. %, less than about 3 wt. %, less than about 2 wt. %,less than about 1 wt. %, less than about 0.5 wt. % or less than about0.1 wt. %. In some embodiments, solvents which can be readily removed byevaporation are not considered as impurities when determining thepercentage purity of a composition or the percentage of impuritiespresent in a composition.

In some embodiments, including any of the foregoing embodiments, thealpha-tocotrienol purity is in the range of from about 80 (A) % to 99.9(A) %, or in the range of from about 85 (A) % to 99.9 (A) %, or in therange of from about 90 (A) % to 99.9 (A) %, or in the range of fromabout 95 (A) % to 99.9 (A) %. In some embodiments, including any of theforegoing embodiments, the alpha-tocotrienol purity is in the range offrom about 80 (A) % to about 99.9 (A) %, or in the range of from about85 (A) % to about 99.9 (A) %, or in the range of from about 90 (A) % toabout 99.9 (A) %, or in the range of from about 95 (A) % to about 99.9(A) %. In some embodiments, including any of the foregoing embodiments,the alpha-tocotrienol purity is greater than 80 (A) %, or greater than85 (A) %, or greater than 90 (A) %, or greater than 91 (A) %, or greaterthan 92 (A) %, or greater than 93 (A) %, or greater than 94 (A) %, orgreater than 95 (A) %, or greater than 96 (A) %, or greater than 97 (A)%, or greater than 98 (A) %, or greater than 99 (A) %, or greater than99.5 (A) %, or greater than 99.9 (A) %. In some embodiments, includingany of the foregoing embodiments, the alpha-tocotrienol purity is morethan about 80 (A) %, or more than about 85 (A) %, or more than about 90(A) %, or more than about 91 (A) %, or more than about 92 (A) %, or morethan about 93 (A) %, or more than about 94 (A) %, or more than about 95(A) %, or more than about 96 (A) %, or more than about 97 (A) %, or morethan about 98 (A) %, or more than about 99 (A) %, or more than about99.5 (A) %, or more than about 99.9 (A) %. In some embodiments,including any of the foregoing embodiments, the impurities in the finalproduct are less than about 20 (A) %, or less than about 15 (A) %, orless than about 10 (A) %, or less than about 5 (A) %, or less than about4 (A) %, or less than about 3 (A) %, or less than about 2 (A) %, or lessthan about 1 (A) %, or less than about 0.5 (A) %, or less than about 0.1(A) %. In some embodiments, including any of the foregoing embodiments,the impurities in the final product comprise tocols or tocol derivativesin the final product and total less than about 5 (A) %, less than about4 (A) %, less than about 3 (A) %, less than about 2 (A) %, less thanabout 1 (A) %, less than about 0.5 (A) % or less than about 0.1 (A) %.In some embodiments, including any of the foregoing embodiments, theimpurities in the final product consist of tocols or tocol derivativesin the final product and total less than about 5 (A) %, less than about4 (A) %, less than about 3 (A) %, less than about 2 (A) %, less thanabout 1 (A) %, less than about 0.5 (A) % or less than about 0.1 (A) %.In some embodiments, solvents which can be readily removed byevaporation are not considered as impurities when determining thepercentage purity of a composition or the percentage of impuritiespresent in a composition.

The feed stream can be any composition comprising the tocotrienol. Incertain embodiments, including any of the foregoing embodiments, thefeed stream can be any composition comprising alpha-tocotrienol. Inadvantageous embodiments, the feed stream is derived from, or the sameas, a product composition of any of the methods described in thesections above. In certain embodiments, including any of the foregoingembodiments, the product composition is adjusted for use as a feedstream. The feed stream generally comprises one or more impuritycompounds in addition to the tocotrienol. In certain embodiments,including any of the foregoing embodiments, the feed stream generallycomprises one or more impurity compounds in addition toalpha-tocotrienol. Impurity compounds are described below.Advantageously, the methods are capable of purifying the tocotrienolfrom the impurity compound or compounds. In certain embodiments,including any of the foregoing embodiments, advantageously, the methodsare capable of purifying alpha-tocotrienol from the impurity compound orcompounds.

The tocotrienol can be any tocotrienol known to those of skill in theart. In certain embodiments, including any of the foregoing embodiments,the tocotrienol can be selected from the group consisting of alphatocotrienol, beta tocotrienol, gamma tocotrienol, and delta tocotrienol.In particular embodiments, the tocotrienol is alpha tocotrienol.

The impurity compounds can be any compound other than the tocotrienol inthe feed stream. In some embodiments, including any of the foregoingembodiments, the impurity compounds can be any compound other thanalpha-tocotrienol in the feed stream. Typically, the impurity compoundsare compounds that the practitioner of skill desires to eliminate fromthe composition. In certain embodiments, including any of the foregoingembodiments, the impurity compounds are selected from tocotrienols otherthan a desired tocotrienol (e.g., alpha-tocotrienol). For instance, inmethods of purifying alpha tocotrienol, the one or more impuritycompounds can be any or all of beta tocotrienol, gamma tocotrienol,delta tocotrienol, and tocopherols (alpha-, beta-, gamma-, ordelta-tocopherol). In addition, the impurity compounds can also beselected from derivatives of the desired tocotrienol (in someembodiments, alpha-tocotrienol) and other impurities, includingnon-tocol impurities, from prior reaction or processing steps or thatmay be present in the starting material.

In some embodiment, including any of the foregoing embodiments, theanalytical method used to determine purity is according to:

Run Parameter Conditions Column HALO RP-Amide, 2.7 μm, 150 * 3 mm Column40° C. Temperature Temperature of 4° C. autosampler Flow 1.4 mL/minMobile Phases Mobile Phase A = 100% water Mobile Phase B = 80%acetonitrile: 20% methanol Gradient Time (min) % A % B 0 60 40 44.0 0100 52.0 0 100 54.0 60 40 61.0 60 40 Sample diluent TetrahydrofuranInjection Volume 5.0 μL Sample Concentra- 0.5 mg/mL tion (NominalTarget) Detection UV: 210 nm (bandwidth 4 nm when using a DAD) Retentiontime alpha-tocotrienol: ca. 26.8 min (RRT: 1.00)

In some embodiments, including any of the foregoing embodiments, analpha tocotrienol-MeOH adduct is one of the impurities in the feedstream used in the first and/or second pass SMB purification(s). In someembodiments, including any of the foregoing embodiments, the alphatocotrienol-MeOH adduct has a relative retention time (RRT) of about0.985 in an UPLC analysis, as measured at a wavelength of 210 nm. Insome embodiments, including any of the foregoing embodiments, the alphatocotrienol-MeOH adduct has the following structure:

In certain embodiments; including any of the foregoing embodiments, thefeed stream contains greater than or equal to about 45 wt %, greaterthan or equal to about 50 wt %, greater than or equal to about 55 wt %,greater than or equal to about 60 wt %, greater than or equal to about65 wt %, greater than or equal to about 70 wt %, greater than or equalto about 75 wt %, or greater than or equal to about 80 wt %, of thealpha-, beta-, gamma-, or delta-tocotrienol, including as compared tothe non-target tocotrienols. In certain embodiments, including any ofthe foregoing embodiments, the feed stream contains about 50 wt %, about51 wt %, about 52 wt %, about 53 wt %, about 54 wt %, about 55 wt %,about 56 wt %, about 57 wt %, about 58 wt %, about 59 wt %, about 60 wt%, about 61 wt %, about 62 wt %, about 63 wt %, about 64 wt %, about 65wt %, about 66 wt %, about 67 wt %, about 68 wt %, about 69 wt %, about70 wt %, about 71 wt %, about 72 wt %, about 73 wt %, about 74 wt %,about 75 wt %, about 76 wt %, about 77 wt %, about 78 wt %, about 79 wt%, about 80 wt %, about 81 wt %, about 82 wt %, about 83 wt %, about 84wt %, or about 85 wt %, of the alpha-, beta-, gamma-, ordelta-tocotrienol, including as compared to the non-target tocotrienols.In certain embodiments, including any of the foregoing embodiments, thefeed stream contains greater than or equal to about 45 wt %, greaterthan or equal to about 50 wt %, greater than or equal to about 55 wt %,greater than or equal to about 60 wt %, greater than or equal to about65 wt %, greater than or equal to about 70 wt %, greater than or equalto about 75 wt %, or greater than or equal to about 80 wt %, of thealpha-tocotrienol. In certain embodiments, including any of theforegoing embodiments, the feed stream contains about 50 wt %, about 51wt %, about 52 wt %, about 53 wt %, about 54 wt %, about 55 wt %, about56 wt %, about 57 wt %, about 58 wt %, about 59 wt %, about 60 wt %,about 61 wt %, about 62 wt %, about 63 wt %, about 64 wt %, about 65 wt%, about 66 wt %, about 67 wt %, about 68 wt %, about 69 wt %, about 70wt %, about 71 wt %, about 72 wt %, about 73 wt %, about 74 wt %, about75 wt %, about 76 wt %, about 77 wt %, about 78 wt %, about 79 wt %,about 80 wt %, about 81 wt %, about 82 wt %, about 83 wt %, about 84 wt%, or about 85 wt %, of the alpha-tocotrienol.

In certain embodiments; including any of the foregoing embodiments, thefeed stream contains greater than or equal to about 60 (A) %, greaterthan or equal to about 65 (A) %, greater than or equal to about 70 (A)%, greater than or equal to about 75 (A) %, greater than or equal toabout 80 (A) %, or greater than or equal to about 85 (A) %, of thealpha-, beta-, gamma-, or delta-tocotrienol, including as compared tothe non-target tocotrienols. In certain embodiments, including any ofthe foregoing embodiments, the feed stream contains about 60 (A) %,about 61 (A) %, about 62 (A) %, about 63 (A) %, about 64 (A) %, about 65(A) %, about 66 (A) %, about 67 (A) %, about 68 (A) %, about 69 (A) %,about 70 (A) %, about 71 (A) %, about 72 (A) %, about 73 (A) %, about 74(A) %, about 75 (A) %, about 76 (A) %, about 77 (A) %, about 78 (A) %,about 79 (A) %, about 80 (A) %, about 81 (A) %, about 82 (A) %, about 83(A) %, about 84 (A) %, or about 85 (A) %, of the alpha-, beta-, gamma-,or delta-tocotrienol, including as compared to the non-targettocotrienols. In certain embodiments, including any of the foregoingembodiments, the feed stream contains greater than or equal to about 60(A) %, greater than or equal to about 65 (A) %, greater than or equal toabout 70 (A) %, greater than or equal to about 75 (A) %, greater than orequal to about 80 (A) %, or greater than or equal to about 85 (A) %, ofthe alpha-tocotrienol. In certain embodiments, including any of theforegoing embodiments, the feed stream contains about 60 (A) %, about 61(A) %, about 62 (A) %, about 63 (A) %, about 64 (A) %, about 65 (A) %,about 66 (A) %, about 67 (A) %, about 68 (A) %, about 69 (A) %, about 70(A) %, about 71 (A) %, about 72 (A) %, about 73 (A) %, about 74 (A) %,about 75 (A) %, about 76 (A) %, about 77 (A) %, about 78 (A) %, about 79(A) %, about 80 (A) %, about 81 (A) %, about 82 (A) %, about 83 (A) %,about 84 (A) %, or about 85 (A) %, of the alpha-tocotrienol.

In some embodiments, the feed stream for SMB purification is preparedusing material made using procedures described in Example 1. In certainembodiments, the material prepared in Example 1 is used directly as thefeed stream. In certain embodiments, the material prepared in Example 1is diluted to a concentration suitable for a feed stream to be used inSMB purification. The feed stream comprises one or more solvents. Incertain embodiments, the feed stream comprises methanol. In certainembodiments, the feed stream comprises the compounds dissolved inmethanol. In some embodiments, the feed stream consists essentially ofthe compounds dissolved in methanol. The compounds can include thedesired tocotrienol (in some embodiments, alpha-tocotrienol) and anyimpurity compounds. In certain embodiments, an oil comprising thedesired tocotrienol (in some embodiments, alpha-tocotrienol) is dilutedin methanol for the feed stream.

In some embodiments, the feed stream for SMB purification is preparedusing material made using procedures described in Example 3. In certainembodiments, the material prepared in Example 3 is used directly as thefeed stream. In certain embodiments, the material prepared in Example 3is diluted to a concentration suitable for a feed stream to be used inSMB purification. The feed stream can comprise one or more solvents. Incertain embodiments, the feed stream comprises acetonitrile, a heptane(in some embodiments, n-heptane), and/or water. In certain embodiments,the feed stream comprises acetonitrile and a heptane (in someembodiments, n-heptane), or water. In certain embodiments, the feedstream comprises acetonitrile. In certain embodiments, the feed streamcomprises the compounds dissolved in acetonitrile, a heptane (in someembodiments, n-heptane), and/or water; and optionally any residualtoluene from the previous step. In certain embodiments, the feed streamconsists essentially of the compounds dissolved in acetonitrile and aheptane (in some embodiments, n-heptane), or water; and optionally anyresidual toluene from the previous step. In certain embodiments, thefeed stream comprises the compounds dissolved in acetonitrile. Incertain embodiments, the feed stream consists essentially of thecompounds dissolved in acetonitrile. In certain embodiments, the feedstream comprises the compounds dissolved in acetonitrile and optionallyany residual toluene from the previous step. In certain embodiments, thefeed stream consists essentially of the compounds dissolved inacetonitrile and optionally any residual toluene from the previous step.The compounds can include the desired tocotrienol (in some embodiments,alpha-tocotrienol) and any impurity compounds. In certain embodiments,an oil comprising the desired tocotrienol (in some embodiments,alpha-tocotrienol) is diluted in acetonitrile for the feed stream.

In some embodiments, the feed stream for SMB purification is preparedusing material made using procedures described in Example 6. In certainembodiments, the material prepared in Example 6 is used directly as thefeed stream. In certain embodiments, the material prepared in Example 6is diluted to a concentration suitable for a feed stream to be used inSMB purification. The feed stream can comprise one or more solvents. Insome embodiments, the feed stream comprises acetonitrile, a heptane (insome embodiments, n-heptane), t-AmOH, and/or water. In some embodiments,the feed stream comprises acetonitrile, a heptane (in some embodiments,n-heptane), and t-AmOH. In some embodiments, the feed stream comprisesacetonitrile and a heptane (in some embodiments, n-heptane). In someembodiments, the feed stream comprises acetonitrile and t-AmOH. In someembodiments, the feed stream comprises acetonitrile and water. In someembodiments, the feed stream comprises acetonitrile. In certainembodiments, the feed stream comprises the compounds dissolved inacetonitrile, a heptane (in some embodiments, n-heptane), t-AmOH, and/orwater. In some embodiments, the feed stream comprises the compoundsdissolved in acetonitrile, a heptane (in some embodiments, n-heptane),and t-AmOH. In some embodiments, the feed stream comprises the compoundsdissolved in acetonitrile and a heptane (in some embodiments,n-heptane). In some embodiments, the feed stream comprises the compoundsdissolved in acetonitrile and t-AmOH. In some embodiments, the feedstream comprises the compounds dissolved in acetonitrile and water. Insome embodiments, the feed stream comprises the compounds dissolved inacetonitrile. In some embodiments, the feed stream consists essentiallyof the compounds dissolved in acetonitrile, a heptane (in someembodiments, n-heptane), t-AmOH, and/or water. In some embodiments, thefeed stream consists essentially of the compounds dissolved inacetonitrile, a heptane (in some embodiments, n-heptane), and t-AmOH. Insome embodiments, the feed stream consists essentially of the compoundsdissolved in acetonitrile and a heptane (in some embodiments,n-heptane). In some embodiments, the feed stream consists essentially ofthe compounds dissolved in acetonitrile and t-AmOH. In some embodiments,the feed stream consists essentially of the compounds dissolved inacetonitrile and water. In some embodiments, the feed stream consistsessentially of the compounds dissolved in acetonitrile. The compoundscan include the desired tocotrienol (in some embodiments,alpha-tocotrienol) and any impurity compounds. In certain embodiments,an oil comprising the desired tocotrienol (in some embodiments,alpha-tocotrienol) is diluted in acetonitrile is provided for the feedstream.

The feed stream can be applied to the SMB apparatus, or the feed streamcan be pre-treated. In certain embodiments, including any of theforegoing embodiments, the feed stream is treated by passing through oneor more silica plugs to remove some impurities. In certain embodiments,including any of the foregoing embodiments, the solvent in the effluentfrom the silica plug treatment is removed to achieve a concentrationsuitable for SMB purification.

The feed stream can comprise the compounds in any concentration deemedsuitable to the practitioner of skill. In particular embodiments, theconcentration is from about 1 to about 200 g/L, from about 5 to about200 g/L, from about 10 to about 200 g/L, from about 20 to about 200 g/L,or from about 25 to about 200 g/L. In particular embodiments, theconcentration is about 38 g/L. In particular embodiments, theconcentration is about 130 g/L. In particular embodiments, theconcentration is about 150 g/L.

In some embodiments, the eluant comprises methanol. In some embodiments,the eluant comprises acetonitrile. In some embodiments, the eluantcomprises acetonitrile and a heptane (in some embodiments, n-heptane).In some embodiments, the eluant comprises acetonitrile and water. Insome embodiments, the eluant comprises acetonitrile, a heptane (e.g.,n-heptane), and t-AmOH. In some embodiments, the eluant comprisesacetonitrile and t-AmOH. In some embodiments, the eluant comprisesacetonitrile, a heptane (e.g., n-heptane), t-AmOH, and/or water.

The stationary phase of the SMB apparatus can be any stationary phasedeemed suitable by the practitioner of skill. In certain embodiments,including any of the foregoing embodiments, the stationary phasecomprises a silica gel. In certain embodiments, including any of theforegoing embodiments, the stationary phase comprises a C₁₈ silica gel.In certain embodiments, including any of the foregoing embodiments, thestationary phase comprises a non silica based packing material(including a polymeric resin).

In particular embodiments, the stationary phase comprises a Chromatorexend-capped C₁₈ hydrophobic silica gel (Fuji). In particular embodiments,the stationary phase comprises a Chromatorex C₁₈ hydrophobic silica gel,and the feed stream comprises methanol. In particular embodiments, thestationary phase comprises a Chromatorex C₁₈ hydrophobic silica gel, andthe feed stream comprises acetonitrile.

In some embodiments, the stationary phase comprises an Ace 10 C18-ARsilica bonded packing material (Advanced Chromatography TechnologiesLtd.). In particular embodiments, the stationary phase comprises an Ace10 C18-AR silica bonded packing material, and the feed stream comprisesacetonitrile. In some embodiments, the eluant comprises acetonitrile andwater.

In some embodiments, the stationary phase comprises a reversed phasegel. In certain embodiments, the stationary phase comprises apolystyrene gel. In certain embodiments, the stationary phase comprisesan MCI CHP20 gel (Mitsubishi). In particular embodiments, the stationaryphase comprises an MCI CHP20/P20 gel (Mitsubishi). In particularembodiments, the stationary phase comprises an MCI CHP20/P20 gel, andthe feed stream comprises acetonitrile. In some embodiments, the eluantcomprises acetonitrile and a heptane (in some embodiments, n-heptane).

In some embodiments, the stationary phase comprises Mac Mod C18 AR 15-20micron packing material. In some embodiments, the stationary phasecomprises Mac Mod C18 AR 15-20 micron packing material, and the feedstream comprises acetonitrile.

The stationary phase can have any size deemed suitable by thepractitioner of skill. In certain embodiments, including any of theforegoing embodiments, the stationary phase has a particle size fromabout 10 microns to about 100 microns. In certain embodiments, includingany of the foregoing embodiments, the stationary phase has a particlesize from about 10 microns to about 25 microns. In certain embodiments,including any of the foregoing embodiments, the stationary phase has aparticle size of about 10 microns, about 15 microns, about 20 microns,about 25 microns, about 30 microns, about 35 microns, about 40 microns,about 50 microns, about 60 microns, about 70 microns, about 80 microns,about 90 microns, or about 100 microns.

The columns of the SMB apparatus can have any size deemed suitable bythe practitioner of skill. In certain embodiments, including any of theforegoing embodiments, the columns have a diameter of about 4.6 mm toabout 1000 mm. In certain embodiments, including any of the foregoingembodiments, the columns have a diameter of about 1 cm to about 10 cm.In certain embodiments, including any of the foregoing embodiments, thecolumns have a length of about 5 cm to about 1 meter. In certainembodiments, including any of the foregoing embodiments, the columnshave a length of about 10 cm to about 1 meter. The practitioner of skillcan increase the column size with the scale of the purification method.

The SMB apparatus can have any number of columns deemed suitable by thepractitioner of skill. In certain embodiments, including any of theforegoing embodiments, the SMB apparatus has at least 3, 4, 5, 6, 7, or8 columns. The practitioner of skill can increase the number of columnswith the scale of the purification method.

The SMB apparatus can be run at any throughput deemed suitable by thepractitioner of skill. In certain embodiments, including any of theforegoing embodiments, the throughput is from about 0.05 kg oil per kgstationary phase per 24 hours to about 3 kg oil per kg stationary phaseper 24 hours. In certain embodiments, including any of the foregoingembodiments, the throughput is at least 0.05 kg oil per kg stationaryphase per 24 hours. In certain embodiments, including any of theforegoing embodiments, the throughput is at least 0.10 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.20 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.25 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.30 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.40 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.50 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.60 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.75 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.90 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 0.95 kg oil per kgstationary phase per 24 hours. In certain embodiments, including any ofthe foregoing embodiments, the throughput is at least 1 kg oil per kgstationary phase per 24 hours. The mass of the oil refers to the oilcomprising tocotrienol that is used to prepare the feed stream.

The SMB apparatus can be run at any pressure deemed suitable to thepractitioner of skill. In certain embodiments, including any of theforegoing embodiments, the SMB apparatus is run at a pressure from about2 bar to about 100 bar. In certain embodiments, including any of theforegoing embodiments, the SMB apparatus is run at a pressure from about15 bar to about 45 bar. In particular embodiments, the SMB apparatus isat a pressure of about 15 bar, about 20 bar, about 25 bar, about 30 bar,about 35 bar, about 40 bar, or about 45 bar.

The SMB flow rates can be any flow rates deemed suitable by thepractitioner of skill. In particular embodiments, the flow rates arefrom about 1 mL/min to about 20 mL min. In certain embodiments,including any of the foregoing embodiments, the flow rates are fromabout 1 mL/min to about 15 mL/min. The flow rates can be the same, oreach flow rate can preferably be adjusted independently by thepractitioner of skill. The flow rates include the eluent flow rate, thefeed flow rate, the extract flow rate, the raffinate flow rate, and therecycle flow rate. A skilled practitioner would know how to scale theflow rates as a function of the columns diameter.

The SMB switch time can be any switch time deemed suitable by thepractitioner of skill. In certain embodiments, including any of theforegoing embodiments, the switch time is from about 1 to about 15minutes. In certain embodiments, including any of the foregoingembodiments, the switch time is from about 1 to about 10 minutes. Inparticular embodiments, the switch time is about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10minutes.

The SMB method can be conducted at any temperature deemed suitable tothe practitioner of skill. In certain embodiments, including any of theforegoing embodiments, the temperature is from about 20° C. to about 55°C. In some embodiments, the temperature is about 20° C., about 25° C.,about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., orabout 55° C. In some embodiments, the temperature is about 25° C. Insome embodiments, the temperature is about 28° C. In some embodiments,the temperature is about 30° C. In some embodiments, the temperature isabout 32° C. In some embodiments, the temperature is about 35° C.

In some embodiments, the feed stream temperature is from about 20° C. toabout 55° C. In some embodiments, the temperature is about 20° C., about25° C., about 30° C., about 35° C., about 40° C., about 45° C., about50° C., or about 55° C. In some embodiments, the feed stream temperatureis about 40° C. In some embodiments, the feed stream temperature isabout 45° C. In some embodiments, the feed stream temperature is about50° C.

In certain embodiments, including any of the foregoing embodiments, theSMB method is run one time. Advantageously, further purification can beachieved by running the SMB method more than once. Accordingly, incertain embodiments, including any of the foregoing embodiments, the SMBmethod is run with one pass, with two passes, with three passes, or withmore than three passes. In some embodiments, the SMB method is run withone pass. In some embodiments, the SMB method is run with two passes. Insome embodiments, the SMB method is run with three passes.

The SMB method can proceed for any time deemed suitable for purificationof the tocotrienol. In particular embodiments, the method proceeds forabout 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours,or about 96 hours. In some embodiments, the method proceeds for about 24hours. In some embodiments, the method proceeds for from about 24 to 36hours. In some embodiments, the method proceeds for about 36 hours. Insome embodiments, the method proceeds for from about 36 to 48 hours. Insome embodiments, the method proceeds for about 48 hours. In someembodiments, the method proceeds for from about 48 to 60 hours. In someembodiments, the method proceeds for about 60 hours. Purificationprogress can be monitored by standard techniques such as thin layerchromatography or high-performance liquid chromatography. In certainembodiments, including any of the foregoing embodiments, the first passpurification removes the late-eluting impurities. In certainembodiments, including any of the foregoing embodiments, the second passremoves the early-eluting impurities. In certain embodiments, includingany of the foregoing embodiments, the first pass purification removesthe early-eluting impurities. In certain embodiments, including any ofthe foregoing embodiments, the second pass removes the late-elutingimpurities. In certain embodiments, including any of the foregoingembodiments, the purification conditions for the second, third, fourth,etc. passes are the same as for the first pass. In certain embodiments,including any of the foregoing embodiments, the purification conditionsfor the second, third, fourth, etc. passes are different than the firstpass.

In some embodiments, including any of the foregoing embodiments, thefeed stream and the raffinate stream are protected from exposure tosunlight and oxygen. In some embodiments, including any of the foregoingembodiments, the feed stream of the first and/or second pass and theraffinate stream resulting from the first and/or second pass areprotected from exposure to sunlight and oxygen.

In some embodiments, including any of the foregoing embodiments, theraffinate collected after the first pass is concentrated before using asthe feed stream in a second pass. In some embodiments, including any ofthe foregoing embodiments, the raffinate collected after the first passis contacted with a silica plug before using as the feed stream in asecond pass. In some embodiments, including any of the foregoingembodiments, the raffinate collected after the first pass is contactedwith a silica plug and the effluent collected and concentrated beforeusing as the feed stream in a second pass. In some embodiments,including any of the foregoing embodiments, the feed stream and theraffinate stream are protected from exposure to sunlight and oxygenduring the concentration step(s).

In certain embodiments, including any of the foregoing embodiments,composition from the raffinate comprises substantially pure tocotrienol.In particular embodiments, the tocotrienol (i.e. alpha, beta, delta, orgamma) is at least about 80 wt % pure, at least about 85 wt % pure, atleast about 90 wt % pure, at least about 91 wt % pure, at least about 92wt % pure, at least about 93 wt % pure, at least about 94 wt % pure, orat least about 95 wt % pure, as a single isomer. In certain embodiments,including any of the foregoing embodiments, composition from theraffinate comprises substantially pure alpha-tocotrienol. In particularembodiments, the alpha-tocotrienol is at least about 80% pure, at leastabout 85 wt % pure, at least about 90 wt % pure, at least about 91 wt %pure, at least about 92 wt % pure, at least about 93 wt % pure, at leastabout 94 wt % pure, or at least about 95 wt % pure, as a single isomer.In certain embodiments, including any of the foregoing embodiments, theweight percent is a measure of the tocotrienol content versus all otherimpurities, including all other tocotrienols. In certain embodiments,including any of the foregoing embodiments, the weight percent is ameasure of the alpha-tocotrienol content versus all other impurities. Incertain embodiments, including any of the foregoing embodiments, theweight percent is measured as a UPLC response at 210 nm compared to apure alpha-tocotrienol standard.

In certain embodiments, including any of the foregoing embodiments,composition from the raffinate comprises substantially pure tocotrienol.In particular embodiments, the tocotrienol (i.e. alpha, beta, delta, orgamma) is at least about 80 (A) % pure, at least about 85 (A) % pure, atleast about 90 (A) % pure, at least about 91 (A) % pure, at least about92 (A) % pure, at least about 93 (A) % pure, at least about 94 (A) %pure, or at least about 95 (A) % pure, as a single isomer. In certainembodiments, including any of the foregoing embodiments, compositionfrom the raffinate comprises substantially pure alpha-tocotrienol. Inparticular embodiments, the alpha-tocotrienol is at least about 80%pure, at least about 85 (A) % pure, at least about 90 (A) % pure, atleast about 91 (A) % pure, at least about 92 (A) % pure, at least about93 (A) % pure, at least about 94 (A) % pure, or at least about 95 (A) %pure, as a single isomer. In certain embodiments, including any of theforegoing embodiments, the area percent is measured as a UPLC responseat 210 nm. In certain embodiments, including any of the foregoingembodiments, the area percent is a measure of the tocotrienol contentversus all other impurities visible at 210 nm, including all othertocotrienols. In certain embodiments, including any of the foregoingembodiments, the area percent is a measure of the alpha-tocotrienolcontent versus all other impurities visible at 210 nm.

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of the American ChemicalSociety or the Journal of Biological Chemistry. Specifically, butwithout limitation, the following abbreviations may be used in theexamples and throughout the specification: AT3 (alpha-tocotrienol); g(grams); mg (milligrams); mL (milliliters); μL (microliters); mM(millimolar); μM (micromolar); Hz (Hertz); MHz (megahertz); mmol(millimoles); h, hr or hrs (hours); min (minutes); MS (massspectrometry); ESI (electrospray ionization); TLC (thin layerchromatography); HPLC (high performance liquid chromatography); UPLC(ultra-high performance liquid chromatography); CDCl₃ (deuteratedchloroform); DMSO-d₆ (deuterated dimethylsulfoxide); MeOH (methanol); wt% (weight percent); A % or (A) % (area percent); and RVE (rotaryevaporator).

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade). All reactions are conducted at room temperature unlessotherwise noted. Synthetic methodologies illustrated herein are intendedto exemplify the applicable chemistry through the use of specificexamples and are not indicative of the scope of the disclosure.

Non-limiting exemplary methods are described in the Examples. It will beappreciated that where typical or preferred process conditions (i.e.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

While the Examples illustrate certain of the diverse methods availablefor use in producing the alpha-tocotrienol compositions, they are notintended to define the scope of reactions or reaction sequences that areuseful in preparing the compositions herein.

Example 1. Preparation of Alpha-Tocotrienol Enriched Compositions fromTocomin® 1. Introduction

Alpha-tocotrienol (AT3) was obtained by the chemical processing ofrefined palm oil (brand name Tocomin 50®). Tocomin 50® is a 50 wt %mixture of tocotrienols and tocopherols (˜12% alpha-tocotrienol; ˜28%combined beta-, gamma-, and delta-tocotrienol; and ˜11%alpha-tocopherol). The remaining mass is thought to be made up ofvarious esters, free acids, carotenoids, squalene, and sterols; theratio of these impurities can vary from lot to lot. Phytosterol contentof the Tocomin 50® in this example was 13.1%.

Briefly, the chemical steps consisted of a Mannich amino-methylationusing 2-methylpiperazine and formaldehyde, followed by a reduction ofthe resultant amino functionality by use of sodium cyanoborohydride. Thereaction mixture was then subjected to a series of workup steps prior topassing through a silica plug. The general synthetic scheme is shownbelow in Scheme 1.

2. Procedure

A 12 L round bottom flask (reactor 1) equipped with mechanical stirrer,temperature probe, reflux condenser, and nitrogen inlet was charged withN-methyl-piperazine (0.996 kg, 9.94 mol) and paraformaldehyde (0.188 kg,6.26 mol). The mixture was heated to 75° C. and stirred for 1.25 h(note: strong exotherm was observed at 55° C.). Tocomin 50® (2.00 kg,2.44 mol) was charged to the flask followed by heating to 105° C. andstirring for 18 h. The reaction was cooled to 75° C. followed byaddition of 2-methyl-2-butanol (3.78 kg).

A 50 L jacketed cylindrical reactor (reactor 2) equipped with amechanical stirrer, temperature probe, nitrogen inlet, reflux condenser,and caustic scrubber was charged with 2-methyl-2-butanol (3.00 kg) andsodium cyanoborohydride (0.784 kg, 12.48 mol). This mixture was heatedto 75° C. and stirred for 1 hour. The contents of reactor 1 weretransferred to reactor 2 over 30 minutes. The contents were heated to105° C. and stirred 28 hours. In process HPLC analysis showedconsumption of the starting material and the contents were cooled to 22°C. Isopropyl acetate (4.26 kg) and water (2.00 kg) were charged to thereactor followed by 10% Na₂HPO₄ (2.00 kg). The biphasic mixture wasstirred for 30 minutes and the resulting solids were filtered. Themixture was then settled for 30 minutes followed by draining the bottomaqueous layer to waste. A second wash of 10% Na₂HPO₄ (2.00 kg) wascharged and the contents were stirred for 30 minutes followed by 30minutes of settling. The bottom aqueous layer was drained to waste. Awash of 10% citric acid (2.50 kg) was charged and the contents werestirred for 30 minutes followed by 30 minutes of settling. The bottomaqueous layer was drained to waste.

The reflux condenser was replaced with a distillation head andcondenser. The jacket temperature was set to 75° C. and vacuum wasapplied. The mixture was distilled to one volume (10 L). Methanol (4.00kg) was charged and the mixture was distilled to 5 L followed by thecharged of methanol (3.00 kg) and distillation to minimum stir volume.The mixture was cooled to −15° C. and Celite® (1.00 kg) was charged.This slurry was filtered and the cake was washed with methanol (2.00kg). The filtrate was returned to reactor 2 (now warmed to 20° C.),diluted with N-methyl-pyrrolidone (4.00 kg), and basified with 30% NaOMein methanol (2.64 kg). This solution was extracted once with 6.00 kgn-heptane and three times with 3.00 kg n-heptane. The n-heptane layerswere discarded. The jacket was set to 10° C. and the basic methanolmixture was acidified to pH 4 with 2M HCl (7.50 kg) while maintaining atemperature below 25° C. Toluene (8.00 kg) was charged, the mixture wasstirred 30 minutes followed by 30 minutes of settle time. The bottomaqueous layer was discarded. The toluene layer was washed three timeswith 10% brine (4.00 kg); the brine layers were discarded.

The jacket was set to 75° C. and the solution was distilled to onevolume under vacuum. The solution was diluted with toluene (5 L). Abench-top filter was charged with silica gel (4.00 kg and packed intoluene). The AT3 solution was passed through the column and 9.00 kg oftoluene was eluted and collected as a series of fractions. The fractionswere analyzed for weight percent AT3 and all fractions (#1-11) over 50wt % AT3 were combined. Weight percent AT3 analysis of the silica plugfractions is shown in Table 3.

TABLE 3 Weight percent AT3 analysis of the silica plug fractions.Fraction Weight Number percent AT3 1 52% 2 53% 3 54% 4 75% 5 53% 6 50% 754% 8 55% 9 54% 10 54% 11 50% 12 37% 13 39%

The combined fractions were then charged to a clean 30 L reactorequipped with mechanical stirrer, temperature probe, distillation headand condenser. The solution was distilled under vacuum to 25% volume(75° C. jacket temperature). Methanol (8.00 kg) was charged and thesolution was distilled to 25% volume (50° C. jacket temperature). Thiswas repeated three times. In process residual solvent GC showed 0.35%toluene content. A portion of the resulting MeOH solution wasconcentrated and the resulting oil was analyzed by UPLC at 52 wt %, 68 A% AT3. The extrapolated total mass of feed oil was 577 g, correspondingto 300 g total AT3 content.

3. Results and Discussion

3.1 Overview

The enrichment process described in section 2 was developed in order toenrich the AT3 content of the Tocomin 50® and remove non-visibleimpurities. The process sought to overcome the shortcomings of theexisting processes.

3.2 Purity of AT3 Throughout the Process Steps

The AT3 content of the oil was tracked throughout the process in Table4. The primary purity analytics being tracked were the UPLC area percentAT3, the weight percent AT3 of the crude oil, and the delta (areapercent minus weight percent). The area percent AT3 represented the UPLCpurity of the feed stream. This measurement accounted for only theimpurities that were visible by UPLC at 210 nm. The weight percent wasmeasured as a UPLC response at 210 nm against a pure AT3 standard(Fluka, #BCBN1012V). This was a measure of the actual AT3 content in thefeed stream versus all other impurities. The delta percentage was ameasure of the non-detectable impurity content in the feed material.

TABLE 4 Tracking AT3 content of the oil throughout the process. Wt %HPLC A % Δ % Process Stage AT3 AT3 (A % − Wt %) Mannich 18 h — 20.7 —Reduction 24 h — 56.5 — Post MeOH 23.8 55.8 32.0 filtration Tol PostNaOMe 30.8 57.7 26.9 treatment Final Enriched 52.0 68.3 16.3 Product

After the Mannich and reduction chemical steps, the area percent AT3increased from 21 A % to 57 A % AT3 by converting the beta-, delta-, andgamma-tocotrienol isomers to the desired alpha-isomer. A 7% enhancementin weight percent was obtained after the basic MeOH treatment, followedby a further 21% improvement from the silica plug to give the finalenriched product.

3.3 Impurity Tracking

It has been previously observed that during the reduction step, there issome regeneration of the unwanted beta-, delta-, and gamma-tocotrienolisomers. These side products are suspected to arise via a mechanismrelated to a reverse-Mannich reaction. The level of these ‘isomers’ areshown in Table 5.

TABLE 5 Tracking the combined β-, δ-, and γ-tocotrienol isomers¹ throughthe process. Process Stage % isomers Isomer/AT3% Mannich 18 h 0.9 4.3Reduction 19 h 8.3 15.0 Reduction 21 h 7.7 13.8 Reduction 24 h 8.4 14.9Final Enriched Product 9.7 14.3 ¹The term ‘isomers’ refers to area ofthe peaks eluting immediately prior to the AT3 in the UPLC chromatogrambetween RRT 0.94 and 0.99. It is suspected that a significant portion ofthese peaks may not be beta-, delta-, or gamma-tocotrienol.Two metrics were used to quantify the amount of these impurities. Thefirst was the overall area percent of the combined impurities relativeto the overall chromatogram. The second was the ‘isomer/AT3’ percentage,which is a ratio of the area counts of the impurities versus the AT3area counts.

Example 2 SMB Purification

Provided herein is an SMB purification of alpha-tocotrienol from a feedstream prepared from Tocomin 50® according to the Example 1.

The chromatography mobile phase was methanol and the stationary phasewas Fuji Chromatorex hydrophobic end-capped C18 in 20 μm diameterspherical particles. The first phase of the SMB purification was tunedto remove any compounds eluting later than alpha tocotrienol. Theprimary identified late-eluting impurity compound was α-tocopherol. Thelate-eluting impurities were isolated in the extract stream of the SMBapparatus, while the alpha tocotrienol and early-eluting impurities wereisolated in the raffinate stream of the SMB apparatus.

Feed for the SMB was prepared by diluting the concentrated oil fromExample 1 in methanol. The total amount of oil (577 g of alphatocotrienol and impurities) was used to calculate the amount of methanolto dilute the supplied feed. The feed was diluted to 38 g/L.

The SMB chromatography system was equipped with 8 individualchromatography columns each 1 cm in diameter and 10 cm in length, allpacked with Fuji Chromatorex C18 hydrophobic end capped 20 μm diameterstationary phase. One cycle through the eight columns was denoted as acomplete cycle. The SMB throughput was 1.39 kg oil/kg stationaryphase/24 hr at 21 bar or 2.32 kg oil/kg stationary phase/24 hr at 35bar.

The raffinate stream was collected as pooled samples and analyzed forarea percent alpha tocotrienol, weight percent alpha tocotrienol, andisomer content. Over 16 hourly samples, the area percent alphatocotrienol varied from 77.7% to 82%, and the weight percent alphatocotrienol varied from 72.1% to 76.7%. The distribution of alphatocotrienol was 99.9% in the raffinate, 0.06% in the extract, and 0.04%in the recycle. After two passes of SMB purification, raffinate withgreater than 85 (A) % purity alpha tocotrienol can be obtained.

Example 3. Preparation of Alpha-Tocotrienol Enriched Compositions fromGold Tri.E™ 70 1. Introduction

Alpha-tocotrienol (AT3) was obtained by the chemical processing ofrefined palm oil (brand name Sime Darby Gold Tri.E™ 70). Gold Tri.E™ 70is a 70% mixture of tocotrienols and tocopherols. The remaining mass isthought to be made up of various esters, free acids, carotenoids,squalene, and sterols; the ratio of these impurities can vary from lotto lot.

Briefly, the chemical steps consisted of a Mannich amino-methylationusing 2-methylpiperazine and formaldehyde, followed by a reduction ofthe resultant amino functionality by use of sodium cyanoborohydride. Thereaction mixture was then subjected to a series of workup steps prior topassing through a silica plug. The general synthetic scheme is shownbelow in Scheme 2.

2. Procedure

A 1 L jacketed reactor (reactor 1) equipped with mechanical stirrer,temperature probe, reflux condenser, and nitrogen inlet was charged withGold Tri.E™ 70 (400.0 g, 0.68 mol) and paraformaldehyde (52.4 g, 1.74mol, 2.56 eq.). N-methyl-piperazine (276.0 g, 2.76 mol, 4.04 eq.) wascharged slowly over 30 minutes in order to control the exotherm <27° C.internal temperature. The mixture was warmed to 75° C. and stirred for30 minutes before warming to 105° C. and stirring for 20 h, after whichtime all starting material was consumed by UPLC. The reaction was cooledto 75° C. followed by addition of 2-methyl-2-butanol (520.0 g, 5.90 mol,8.65 eq.).

A 5 L jacketed reactor (reactor 2) equipped with a mechanical stirrer,temperature probe, nitrogen inlet, reflux condenser, and causticscrubber was charged with 2-methyl-2-butanol (840.0 g, 9.53 mol, 13.98eq.) and sodium cyanoborohydride (220.0 g, 3.50 mol, 5.13 eq.). Reactor2 was heated to 75° C. and stirred for 1 hour. The contents of reactor 1were transferred to reactor 2 over 30 minutes. The contents of reactor 2were heated to 107° C. and stirred 28 hours, after which time in-processUPLC analysis showed consumption of the starting material and thecontents were cooled to 20° C. Water (400.0 g) was charged and a mildexotherm was observed (+6° C.). iPrOAc (852.0 g) was charged and themixture was warmed to 60° C. for 1 h in order to dissolve the majorityof precipitated solids. The layers were settled for 30 minutes and thebottom aqueous phase was drained (aq: 616 g, org: 3.9 L). A secondportion of H₂O was charged (400 g) and the mixture was stirred at 60° C.for 30 min. then settled for 30 min. The bottom aqueous phase wasdrained (aq: 460 g, org: 3.6 L). The reaction mixture was cooled to 20°C. and a 30 wt % solution of citric acid/H₂O (480 g, 0.75 mol, 1.10 eq.)was charged. The mixture was stirred 30 min. then settled for 30 min.,after which time the aqueous phase was drained (aq: 658 g, org: 3.3 L).Reactor 2 was set to distillation mode and the jacket was set to 70° C.The volatile components were removed by vacuum distillation until themixture became a thick oil. MeOH (600 g) was charged and then distilleduntil the mixture became a thick oil. The reactor was cooled to 20° C.,whereupon MeOH (800 g) and celite (200 g) were charged and the slurrywas cooled to −15° C. The mixture was stirred for 1 h at −15° C. and theprecipitate was removed by filtration, followed by a cake wash with −15°C. MeOH (400 g).

The filtrate was returned to reactor 2, cooled to 0° C., diluted withN-methyl-pyrrolidone (800 g), and basified with 30% NaOMe in methanol(528 g) (mild exotherm+9° C.). To this solution was charged n-heptane(800 g) at 0° C. (total volume: 4.1 L), stirred 30 min, settled 1 h. Thetop heptane phase was removed (hept: 425 mL, MeOH: 3.7 L) and a secondportion of n-heptane (600 g) was charged at 0° C. (total volume: 4.5 L),stirred 30 min, settled 30 min, and the heptane phase was removed (hept:940 mL, MeOH: 3.5 L). The extraction with n-heptane was repeated twomore times, discarding the heptane phase each time. The organic MeOHphase was returned to reactor 1 at 0° C. and the basic methanol mixturewas acidified to pH 7 with 2M HCl (1510 g) while maintaining atemperature below 25° C. The jacket temperature was adjusted to 20° C.,and toluene (1600 g) was charged and the mixture was stirred 30 minutesfollowed by 1 h of settle time. The bottom aqueous layer was discarded.The toluene layer was warmed to 60° C. and 5% brine was added. Themixture was stirred 30 min and settled 12 h in order to achieve a cleanphase split. The brine phase was discarded and a second portion of 5%brine was charged (800 g) at 60° C. The phases were stirred for 30 minand then settled 12 h, at which time a clean phase split was achievedand the brine phase was discarded.

The organic phase was returned to reactor 1 (total volume 3.05 L) andthe jacket was set to 75° C. The solution was distilled under reducedpressure to the desired volume (1.8 L). The toluene solution was loadedonto a silica column (800 g SiO₂, packed in toluene) and the lightyellow eluent (560 g) was discarded. The column was and washed withtoluene (2500 g) and the eluent was collected after 350 g toluene hadeluted. A total mass of 2128 g eluent were collected and concentrated toa viscous orange oil. The oil was blown dry under a stream of nitrogen.A total of 180.99 g oil was collected, with 66.5 A % and 59.3 wt % AT3by UPLC analysis. Once corrected for weight percent, a total of 107.33 gAT3 was isolated, resulting in a 49.97% theoretical AT3 yield (based ofthe 53.7 wt % tocotrienol content of the starting material). Thematerial was used as the feed in for the 2-pass SMB purification.

Example 4. Waste Stream Analysis

All waste streams generated during the bulk feed preparation in Example3 were isolated and analyzed for AT3 content (AFC-704-051). The goal ofthis analysis was to determine if any processing steps resulted in largelosses of AT3. The results of this analysis are tabulated in the form ofAT3 mass (grams) and AT3 loss as a percentage of theoretical AT3 yield(Table 6). The mass balance data indicates that the processing steps arequite efficient, resulting in a total of 3.3 g lost to waste streams, or1.65% AT3 yield loss.

TABLE 6 AT3 losses to waste streams throughout process AT3 mass Yieldlost Step (g) (%) Aqueous quench #1 0.02 0.01 Aqueous quench #2 0 0Citric acid wash 0 0 Hept extract #1 0.07 0.04 Hept extract #2 0.23 0.11Hept extract #3 0.46 0.23 Hept extract #4 0.25 0.12 Neutralized aq layer0.14 0.07 Brine wash #1 0.31 0.15 Brine wash #2 0 0 Column wash 1.830.92 Total losses 3.30 1.65

3. Results and Discussion

3.1 Overview

The enrichment process described in section 2 was developed in order toenrich the AT3 content of the Gold Tr.E™ 70 and remove non-visibleimpurities. The process sought to overcome the shortcomings of theexisting processes.

Example 5. SMB Purification

Provided herein is an SMB purification of alpha-tocotrienol from a feedstream prepared from Gold Tr.E™ 70 according to Example 3. The startingalpha-tocotrienol purity was 66.5 area percent and 59.3 weight percent.

The chromatography mobile phase was acetonitrile and the stationaryphase was Mitsubishi MCI gel CHP20/P20 20 μm diameter particles. Thefirst phase of the SMB purification was tuned to remove any compoundseluting later than alpha-tocotrienol. The primary identifiedlate-eluting compound was α-tocopherol. The late-eluting impurities wereisolated in the extract stream of the SMB apparatus, while thealpha-tocotrienol and early-eluting impurities were isolated in theraffinate stream of the SMB apparatus.

Feed for the SMB was prepared by diluting the concentrated oil fromExample 3 in acetonitrile. The oil (167.44 g of alpha-tocotrienol andimpurities) was diluted to approx. 150 g/L in acetonitrile,respectively.

The SMB chromatography system was equipped with 6 individualchromatography columns each 1 cm in diameter and 10 cm in length. Onecycle through the six columns was denoted as a complete cycle. For thefirst pass, the SMB throughput was 0.24 kg oil/kg stationary phase/24 hrat approx. 36 to approx. 40 bar. The raffinate was collected, and 47 galpha-tocotrienol was collected with a purity of 90.8 area percent.

The feed for the second pass was prepared by concentrating the recoveredraffinate down to the desired volume. Prior to the final evaporation,the mass of oil was estimated based on the gravimetrically determinedconcentration of oil in the raffinate from the first pass. A total of47.91 g of oil was concentrated to 130 g/L. After the second pass of SMBpurification at a throughput of 0.08 kg oil/kg stationary phase/24 hr,the raffinate was collected. 28.8 g alpha-tocotrienol was recovered witha purity of 90.8 area percent.

Example 6. Preparation of Alpha-Tocotrienol Enriched Compositions fromNatural Palm Oil (TC84, Davos Life Science) 1. Introduction

Alpha-tocotrienol (AT3) was obtained by the chemical processing ofnatural palm oil (brand name Davos Life Science TC84). TC84 contains 84A % of a mixture of tocotrienols and tocopherols, including AT3, beta-,gamma-, and delta-tocotrienol and the corresponding tocopherols. Theremaining mass is thought to be made up of various esters, free acids,carotenoids, squalene, and sterols; the ratio of these impurities canvary from lot to lot.

Briefly, the chemical steps comprise a two-step telescopic process toconvert beta-, gamma-, and delta-tocotrienol to AT3. Step 1 includes aMannich reaction conducted under neat condition, followed by azeotropicdistillation to remove water byproduct. The intermediates produced inStep 1 are then subjected to reductive cleavage using sodiumcyanoborohydride in Step 2. The resulting enriched AT3 crude ispartially purified by a plug flow silica gel filtration. Finalpurification is performed with an SMB chromatography separation processdescribed in Example 7. The general synthetic scheme is shown below inScheme 3.

2. Procedure

Overview.

The first step in the enrichment process is the Mannich reaction, whichconverts the beta-, gamma- and delta-tocotrienols to themono-methyl-(N-methylpiperazinyl) intermediate in about 2-3 hours, atbatch temperature of about 75° C. to 80° C. under neat condition usingparaformaldehyde and N-methylpiperazine. A second Mannich reactionconverts delta-tocotrienol mono-methyl-(N-methylpiperazinyl) to the thebis-methyl-(N-methylpiperazinyl) intermediate. This second Mannichreaction takes place at a batch temperature of about 110° C. to 120° C.and for a duration of about 12-16 hours. The first Mannich reaction canbe completed, with or without the removal of water, in about 2-3 hours.However, since the batch temperature is limited by the refluxingtemperature of the solvent (t-AmOH, about 90-104° C.), it is preventedfrom reaching a temperature of about 110-120° C. for the second Mannichreaction to convert. Therefore, in the presence of solvent such ast-AmOH (bp=105° C.), water is azeotropically removed during formation ofMannich reaction products. Accordingly, the Mannich reaction isperformed under neat conditions in two steps: (1) the first Mannichreaction is carried out at 75-80° C. in about 2-3 hours, and (2) thesecond Mannich reaction is carried out at 110-115° C. in about 10-16hours. The water by-product produced during the Mannich reaction isremoved by azeotropic distillation in the presence of t-AmOH prior tothe reduction reaction. Partial removal of amino compounds (e.g.,N-methylpiperazine) can also occur during azeotropic distillation.

Mannich Reaction.

Paraformaldehyde was charged to a 10 L reactor (reactor 1) with jackettemperature set at 35° C. Warm TC84 (760 g, about 40-45° C.) was pouredinto the reactor from the aluminum container, followed byN-methylpiperazine. The batch was held at about 75-80° C. (jackettemperature=80° C.) for 3 hours. The jacket temperature was subsequentlyset to 120° C., and the Mannich reaction was complete after 20 hourswith no trace of delta-/beta-/gamma-tocotrienol detected. The level ofdelta-tocotrienol-methyl(methylpiperazine) was less than 5 A % (about1.3 A %). Two azeotropic distillations, under vacuum at 75-100° C., wereperformed with t-AmOH; the final Mannich product solution in t-AmOH hasa Karl Fischer titration value (KF) of 9.2 ppm.

Reduction.

In a second reactor (reactor 2), 418 g of NaCN(BH₃) was heated at about75-80° C. for 1 hour in t-AmOH (1596 g) before the distillate containingthe Mannich product in t-AmOH was charged. The jacket temperature wasset at 115° C., allowing the batch internal temperature to reach about104° C. At 26 hours, liquid and white solids were observed outside thevessel, collecting around one stopper, indicating the possibility ofinsufficient condenser cooling. An additional 248 g of NaCN(BH₃) wascharged from a new container, and the reaction was continued for 16hours until analysis indicated that reduction was complete (i.e., notrace of intermediate tocotrienol-Mannich product observed). However, itis possible to carry out the reduction reaction without additionalcharging of NaCN(BH₃). Accordingly, in some embodiments, the Mannichproduct (in t-AmOH) and NaCN(BH₃) are mixed and held at a temperature ofabout 110-115° C. for about 12-24 hours in order for the reductionreaction to proceed to completion.

Quench and Wash.

After completion of the reduction reaction, the batch was cooled to20-25° C. and quenched with water (about 2S, or twice the amount ofstarting material), followed by addition of heptane (about 1S-2S, whereS denotes a weight equivalent of starting material). The quenched batchwas held for two hours at 20-25° C., and insoluble solids were filteredout. The solids were rinsed with heptane and t-AmOH, and the filtratefrom each filtration step was combined and charged back to a cleanreactor and allowed to phase separate. The lower aqueous phase wasdrained, and a water wash of the remaining organic phase was carriedout. The lower aqueous phase was again drained, and aqueous citric acid(10%, w/w) was used to wash the remaining organic phase. The loweraqueous phase was drained, leaving an organic phase with a pH of about6.5. The organic phase was then distilled with t-AmOH:heptane in a 1:99volumetric ratio to a desired volume (about 2 L) in heptane.

Silica Filtration.

The heptane solution (temperature about 30-35° C.) was loaded onto asilica column (380 g SiO₂, 12 cm diameter×7.6 cm height) at roomtemperature and allowed to drain until the column was dry. The columnwas rinsed with 600 mL heptane from a heptane reactor rinse and combinedwith the eluate, making up the first column fraction. Three subsequentfractions were subsequently collected from the column, with the firsttwo fractions containing 1.5 L of t-AmOH:heptane in a 1:99 volumetricratio and the last fraction containing 1.5 L of 100% t-AmOH. The contentof each fraction was estimated. Fraction 1 contained 65.9 A % of AT3(about 640 g of crude AT3), and Fraction 2 contained 68.3 A % AT3 (about14 g of crude AT3). Table 5 illustrates the contents of each fractioncollected from the SiO₂ column.

TABLE 7 SiO₂ filtration fractions Mass vs. starting AT3 Frac- BulkEstimated material content tion Description weight content (760 g)(UPLC) Feed Concentrated N/A 57.9 A % solution before SiO₂ separation 12 L batch + 1860 g  639.8 g  0.84 S 65.9 A % 0.6 L heptane rinse 2 1.5 Lheptane: 856 g 13.6 g 0.02 S 68.3 A % t-AmOH (99:1) 3 1.5 L heptane: 878g  8.2 g 0.01 S 63.0 A % t-AmOH (99:1) 4 1.5 L heptane: 868.4 g  19.6 gN/A 18.2 A % t-AmOH (99:1) 5 1.5 L t-AmOH 1015.7 g   99.5 g N/A N/D

Fractions 1 and 2 were combined, and the fractions were distilled toreduce the volume to about 2V, followed by distillation withacetonitrile to provide a concentrated solution in acetonitrile. Thefinal material was a suspension in acetonitrile with an oilconcentration of about 280 g/L; this material was diluted withacetonitrile to about 60 g/L and used as the feed for SMB purificationin Example 7.

Example 7. SMB Purification

Provided herein is an SMB purification of alpha-tocotrienol from a feedstream prepared from natural palm oil (e.g., TC84, Davos Life Science)according to Example 6. The starting material was a suspension ofalpha-tocotrienol in acetonitrile with an oil concentration of about 280g/L.

Testing of the columns used in the SMB unit was conducted with a sampleof the feed stream prepared according to Example 6. Each column was 1 cmdiameter×10 cm length and packed with Mac Mod C₁₈ AR 15-20 micronstationary phase. The columns were each tested with three analyticalinjections of a diluted sample of feed using acetonitrile as a mobilephase. The average retention time of alpha-tocotrienol was 2.856(±0.056) minutes.

The feed stream starting material suspension (280 g/L) was diluted inacetonitrile to about 60 g/L and separated using Mac Mod C₁₈ AR 15-20 μmstationary phase. The column configuration was 1-2-2-1. The chemicallytreated feed solution was subjected to two passes through the mini SMB.The first pass was used to remove the impurities that elute sooner thanthe AT3. Under these conditions the AT3 is recovered in the extractstream. A portion of the very late eluting non-UV visible impurities mayalso be reduced in this first pass; these very late eluting impuritiesbecome distributed throughout the whole column set. The second passremoves the impurities that elute later than the AT3. Under theseconditions the AT3 is recovered in the raffinate.

Feed for the SMB was prepared by diluting the concentrated oil fromExample 6 in acetonitrile. The oil (containing alpha-tocotrienol andimpurities) was diluted to approximately 60 g/L in acetonitrile, whichcontained about 31 g/L AT3. This feed was held at a temperature of about45° C. so that the components would remain in solution.

The SMB chromatography system was equipped with 6 individualchromatography columns each 1 cm in diameter and 10 cm in length. Onecycle through the six columns was denoted as a complete cycle. For thefirst pass, the SMB throughput was 0.99 kg oil/kg stationary phase/24 hrat about 35 bar operating pressure. The extract was collected, and 27.8g alpha-tocotrienol was collected with a purity of 67.2 area percent.This corresponded to an AT3 recovery of 95% after the first pass. Table6 illustrates the parameters of an exemplary first pass through the SMBunit.

TABLE 6 SMB Parameters, First Pass Feed concentration RP-UPLC Feed (g/L)AT3 Zone 1 Extract Feed concentration (pure AT3 Period purity/ (mL/min)(mL/min) (mL/min) (g/L) basis) (min) Extract 14.63 8.40 0.170 60.0 31.01.66 76.9%

For the second pass through the SMB unit, the switch time was changed inorder to push the AT3 into the raffinate stream. Under these conditions,the late eluting impurities are removed in the extract stream. Aproductivity of 0.72 kg oil/kg stationary phase/24 hr was achieved at 35bars operating pressure. An amount of 14.92 grams of alpha-tocotrienolwas recovered with a purity of 97.2 area percent. This corresponded toan AT3 recovery of 93% after the second pass. Table 7 illustrates theparameters of an exemplary second pass through the SMB unit.

TABLE 7 SMB Parameters, Second Pass Feed concentration RP-UPLC (g/L) AT3Zone 1 Extract Feed (pure AT3 Period purity/ (mL/min) (mL/min) (mL/min)basis) (min) Extract 14.63 8.40 0.170 31.0 1.9 97.23%

The productivity of pure AT3 in the combined first and second passes isequivalent to 0.51 kg AT3/kg stationary phase/24 hr.

The columns in the SMB unit were cleaned with isopropanol between thefirst and second passes. However, this step may become optional once thecondition of the feed stream is optimized to maintain the components insolution.

In addition, while removal of the early eluting impurities was performedprior to removal of late eluting impurities, the two passes can bereversed if needed. Accordingly, in some embodiments, a method ofpurifying a tocotrienol by SMB chromatography is provided, whereinremoval of the late eluting impurities takes place during the first passunder conditions provided, e.g., in Table 6, and removal of the earlyeluting impurities takes place during the second pass under conditionsprovided, e.g., in Table 5.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference. While theclaimed subject matter has been described in terms of variousembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the claimed subject matter is limited solely by the scope ofthe following claims, including equivalents thereof.

What is claimed is:
 1. A method of purifying a tocotrienol from a feedsolution comprising the steps of: a. passing the feed solution through achromatographic process comprising a stationary phase and a mobile phasestream, wherein said feed solution comprises the tocotrienol and one ormore impurity compounds; and wherein the mobile phase stream comprisesone or more solvents selected from acetonitrile, ethanol, and isopropylalcohol; b. operating the chromatographic process as a simulated movingbed (SMB) process under conditions effective to purify the tocotrienolfrom at least one impurity compound; c. collecting a stream from the SMBapparatus, wherein said stream comprises the purified tocotrienol; andd. repeating steps a.-c.; wherein the stream from the SMB apparatus isconcentrated before its use as the feed solution in repeating stepsa.-c.
 2. The method of claim 1, wherein the stream comprising thepurified tocotrienol is an extract stream.
 3. The method of claim 1,wherein the stream comprising the purified tocotrienol is a raffinatestream.
 4. The method of claim 1, wherein the stream comprising thepurified tocotrienol is the extract stream in a first pass, and whereinthe stream comprising the purified tocotrienol is the raffinate streamin a second or subsequent pass.
 5. The method of claim 1, wherein thestream comprising the purified tocotrienol is the raffinate stream in afirst pass, and wherein the stream comprising the purified tocotrienolis the extract stream in a second or subsequent pass.
 6. The method ofclaim 1, wherein the tocotrienol is selected from the group consistingof alpha-tocotrienol, beta-tocotrienol, delta-tocotrienol, andgamma-tocotrienol.
 7. The method of claim 6, wherein the tocotrienol isalpha-tocotrienol.
 8. The method of claim 1, wherein the stationaryphase is selected from the group consisting of silica gel,functionalized silica gel, reverse phase gel, or chiral phase gel. 9.The method of claim 1, wherein the stationary phase has a particle sizeof about 2 to about 300 μm.
 10. The method of claim 1, wherein themobile phase stream further comprises one or more solvents selected fromthe group consisting of: water, t-AmOH, methanol, n-proposal, butanol,ethyl acetate, isopropyl acetate, MtBE, diethyl ether, fluorinatedsolvents, alkanes, hexanes, n-hexane, heptanes, n-heptane,methyl-cyclopentane, pentane, methyl-cyclohexane, cyclohexane, toluene,and CO₂.
 11. The method of claim 1, wherein the mobile phase streamcomprises acetonitrile.
 12. The method of claim 1, wherein the mobilephase stream comprises ethanol or isopropyl alcohol.
 13. The method ofclaim 1, wherein the alpha-tocotrienol in the feed stream is preparedaccording to a method for making an alpha-tocotrienol-enriched tocolmixture, comprising: (a) contacting a tocol mixture with anamino-alkylating agent, wherein the tocol mixture comprises at least onenon-alpha-tocotrienol, at least one non-tocol, optionallyalpha-tocotrienol, and optionally one or more tocopherols, whereby theat least one non-alpha-tocotrienol is amino-alkylated; (b) reducing theamino-alkylated non-alpha-tocotrienols to alpha-tocotrienol with areducing agent; (c) removing one or more waxy impurities from themixture; (d) contacting the mixture with an agent that binds one or morepolar impurities; and (e) removing the agent that binds one or morepolar impurities.
 14. The method of claim 1, wherein a final productstream comprising purified tocotrienol comprises the tocotrienol with atleast about 90 (A) % purity.
 15. The method of claim 14, wherein thefinal product stream comprising purified tocotrienol is concentrated toa tocotrienol concentration of at least about 90 wt %.
 16. The method ofclaim 1, wherein the chromatographic process comprises 2 to 30 columnsserially connected.
 17. The method of claim 1, wherein thechromatographic process is operated at a rate of about 0.05 to about 5kg feed stream per kg stationary phase per 24 hours.
 18. The method ofclaim 1, wherein the chromatographic process is operated at a pressureof about 2 bar to about 100 bar.
 19. The method of claim 1, wherein thechromatographic process is operated at a temperature of about 10° C. toabout 50° C.
 20. The method of claim 1, wherein the stationary phasecomprises a C₁₈ silica gel.
 21. The method of claim 20, wherein thestationary phase has a particle size from about 10 μm to about 25 μm.22. The method of claim 3, wherein the raffinate stream is collected andtocotrienol-containing fractions are subjected to a method for making analpha-tocotrienol enriched tocol mixture, comprising: (a) contacting atocol mixture with an amino-alkylating agent, wherein the tocol mixturecomprises at least one non-alpha-tocotrienol, at least one non-tocol,optionally alpha-tocotrienol, and optionally one or more tocopherols,whereby the at least one non-alpha-tocotrienol is amino-alkylated; (b)reducing the amino-alkylated non-alpha-tocotrienols to alpha-tocotrienolwith a reducing agent; (c) removing one or more waxy impurities from themixture; (d) contacting the mixture with an agent that binds one or morepolar impurities; and (e) removing the agent.
 23. The method of claim10, wherein the SMB method is run with two passes, with three passes, orwith more than three passes.